Diagnostics and therapeutics for diseases associated with neuromedin u1 receptor (nmu1)

ABSTRACT

The invention provides a human NMU1 which is associated with the hematological diseases, cardiovascular diseases, disorders of the peripheral and central nervous system, inflammation diseases, cancer diseases, and disorders of the liver. The invention also provides assays for the identification of compounds useful in the treatment or prevention of hematological diseases, cardiovascular diseases, disorders of the peripheral and central nervous system, inflammation diseases, cancer diseases, and disorders of the liver. The invention also features compounds which bind to and/or activate or inhibit the activity of NMU1 as well as pharmaceutical compositions comprising such compounds.

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of molecular biology; moreparticularly, the present invention relates to nucleic acid sequencesand an amino acid sequences of a human NMU1 and its regulation for thetreatment of hematological diseases, cardiovascular diseases, disordersof the peripheral and central nervous system, inflammation diseases,cancer diseases, and disorders of the liver in mammals.

BACKGROUND OF THE INVENTION

G-Protein Coupled Receptors

NMU1 is a seven transmembrane G protein coupled receptor (GPCR) [[Shanet al., 2000] [Raddatz et al., 2000]]. Many medically significantbiological processes are mediated by signal transduction pathways thatinvolve G-proteins [Lefkowitz, (1991)]. The family of G-protein coupledreceptors (GPCRs) includes receptors for hormones, neurotransmitters,growth factors, and viruses. Specific examples of GPCRs includereceptors for such diverse agents as dopamine, calcitonine, adrenergichormones, endotheline, cAMP, adenosine, acetylcholine, serotonine,histamine, thrombin, kinine, follicle stimulating hormone, opsins,endothelial differentiation gene-1, rhodopsins, odorants,cytomegalovirus, G-proteins themselves, effector proteins such asphospholipase C, adenyl cyclase, and phosphodiesterase, and actuatorproteins such as protein kinase A and protein kinase C.

GPCRs possess seven conserved membrane-spanning domains connecting atleast eight divergent hydrophilic loops. GPCRs, also known as seventrans-membrane, 7TM, receptors, have been characterized as includingthese seven conserved hydrophobic stretches of about 20 to 30 aminoacids, connecting at least eight divergent hydrophilic loops. Most GPCRshave single conserved cysteine residues in each of the first twoextracellular loops, which form disulfide bonds that are believed tostabilize functional protein structure. The seven transmembrane regionsare designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 is beingimplicated with signal transduction. Phosphorylation and lipidation(palmitylation or farnesylation) of cysteine residues can influencesignal transduction of some GPCRs. Most GPCRs contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several GPCRs, such as the beta-adrenergicreceptor, phosphorylation by protein kinase A and/or specific receptorkinases mediates receptor desensitization.

For some receptors, the ligand binding sites of GPCRs are believed tocomprise hydrophilic sockets formed by several GPCR transmembranedomains. The hydrophilic sockets are surrounded by hydrophobic residuesof the GPCRs. The hydrophilic side of each GPCR transmembrane helix ispostulated to face inward and form a polar ligand binding site. TM3 isbeing implicated with several GPCRs as having a ligand binding site,such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine, andTM6 or TM7 phenylalanines or tyrosines also are implicated in ligandbinding.

GPCRs are coupled inside the cell by heterotrimeric G-proteins tovarious intracellular enzymes, ion channels, and transporters. DifferentG-protein alpha-subunits preferentially stimulate particular effectorsto modulate various biological functions in a cell. Phosphorylation ofcytoplasmic residues of GPCRs is an important mechanism for theregulation of some GPCRs. For example, in one form of signaltransduction, the effect of hormone binding is the activation of theenzyme, adenylate cyclase, inside the cell. Enzyme activation byhormones is dependent on the presence of the nucleotide GTP. GTP alsoinfluences hormone binding. A G-protein connects the hormone receptor toadenylate cyclase. G-protein exchanges GTP for bound GDP when activatedby a hormone receptor. The GTP-carrying form then binds to activatedadenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-proteinitself, returns the G-protein to its basal, inactive form. Thus, theG-protein serves a dual role, as an intermediate that relays the signalfrom receptor to effector, and as a clock that controls the duration ofthe signal.

Over the past 15 years, nearly 350 therapeutic agents targeting 7TMreceptors have been successfully introduced into the market. Thisindicates that these receptors have an established, proven history astherapeutic targets. Clearly, there is a need for identification andcharacterization of further receptors which can play a role inpreventing, ameliorating, or correcting dysfunctions or diseasesincluding, but not limited to, infections such as bacterial, fungal,protozoan, and viral infections, particularly those caused by HIVviruses, cancers, allergies including asthma, cardiovascular diseasesincluding acute heart failure, hypotension, hypertension, anginapectoris, myocardial infarction, hematological diseases, genito-urinarydiseases including urinary incontinence and benign prostate hyperplasia,osteoporosis, and peripheral and central nervous system disordersincluding pain, Alzheimer's disease and Parkinson's disease.

TaqMan-Technology/Expression Profiling

TaqMan is a recently developed technique, in which the release of afluorescent reporter dye from a hybridisation probe in real-time duringa polymerase chain reaction (PCR) is proportional to the accumulation ofthe PCR product. Quantification is based on the early, linear part ofthe reaction, and by determining the threshold cycle (CT), at whichfluorescence above background is first detected.

Gene expression technologies may be useful in several areas of drugdiscovery and development, such as target identification, leadoptimization, and identification of mechanisms of action. The TaqMantechnology can be used to compare differences between expressionprofiles of normal tissue and diseased tissue. Expression profiling hasbeen used in identifying genes, which are up- or down regulated in avariety of diseases. An interesting application of expression profilingis temporal monitoring of changes in gene expression during diseaseprogression and drug treatment or in patients versus healthyindividuals. The premise in this approach is that changes in pattern ofgene expression in response to physiological or environmental stimuli(e.g., drugs) may serve as indirect clues about disease-causing genes ordrug targets. Moreover, the effects of drugs with established efficacyon global gene expression patterns may provide a guidepost, or a geneticsignature, against which a new drug candidate can be compared.

NMU1

The nucleotide sequence of NMU1 is accessible in public databases by theaccession number AF272362 and is given in SEQ ID NO: 1. The amino acidsequence of GPR NMU1 is depicted in SEQ ID NO: 2.

NMU1 is described as a receptor of the neuropeptide neuromedin U [Shanet al., 2000]. The receptor NMU1 is published in WO-200140797,WO-200125269, WO-200144297. The expression of NMU1 in brain—but not inspecific brain tissues—was previously described [Raddatz et al., 2000].NMU1 shows the highest homology (81%) to the murine receptor FM-3 asshown in example 1.

SUMMARY OF THE INVENTION

The invention relates to novel disease associations of NMU1 polypeptidesand polynucleotides. The invention also relates to novel methods ofscreening for therapeutic agents for the treatment of hematologicaldiseases, cardiovascular diseases, disorders of the peripheral andcentral nervous system, inflammation diseases, cancer diseases, anddisorders of the liver in a mammal. The invention also relates topharmaceutical compositions for the treatment of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver in a mammals comprising a NMU1 polypeptide, a NMU1 polynucleotide,or regulators of NMU1 or modulators of NMU1 activity. The inventionfurther comprises methods of diagnosing hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver in a mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a NMU1 receptor polynucleotide(SEQ ID NO: 1).

FIG. 2 shows the amino acid sequence of a NMU1 receptor polypeptide (SEQID NO: 2).

FIG. 3 shows the nucleotide sequence of a primer useful for theinvention (SEQ ID NO:3).

FIG. 4 shows the nucleotide sequence of a primer useful for theinvention (SEQ ID NO: 4).

FIG. 5 shows a nucleotide sequence useful as a probe to detect proteinsof the invention (SEQ ID NO: 5).

DETAILED DESCRIPTION OF THE INVENTION

Definition of Terms

An “oligonucleotide” is a stretch of nucleotide residues which has asufficient number of bases to be used as an oligomer, amplimer or probein a polymerase chain reaction (PCR). Oligonucleotides are prepared fromgenomic or cDNA sequence and are used to amplify, reveal, or confirm thepresence of a similar DNA or RNA in a particular cell or tissue.Oligonucleotides or oligomers comprise portions of a DNA sequence havingat least about 10 nucleotides and as many as about 35 nucleotides,preferably about 25 nucleotides.

“Probes” may be derived from naturally occurring or recombinant single-or double-stranded nucleic acids or may be chemically synthesized. Theyare useful in detecting the presence of identical or similar sequences.Such probes may be labeled with reporter molecules using nicktranslation, Klenow fill-in reaction, PCR or other methods well known inthe art. Nucleic acid probes may be used in southern, northern or insitu hybridizations to determine whether DNA or RNA encoding a certainprotein is present in a cell type, tissue, or organ.

A “fragment of a polynucleotide” is a nucleic acid that comprises all orany part of a given nucleotide molecule, the fragment having fewernucleotides than about 6 kb, preferably fewer than about 1 kb.

“Reporter molecules” are radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents which associate with aparticular nucleotide or amino acid sequence, thereby establishing thepresence of a certain sequence, or allowing for the quantification of acertain sequence.

“Chimeric” molecules may be constructed by introducing all or part ofthe nucleotide sequence of this invention into a vector containingadditional nucleic acid sequence which might be expected to change anyone or several of the following NMU1 characteristics: cellular location,distribution, ligand-binding affinities, interchain affinities,degradation/turnover rate, signaling, etc.

“Active”, with respect to a NMU1 polypeptide, refers to those forms,fragments, or domains of a NMU1 polypeptide which retain the biologicaland/or antigenic activity of a NMU1 polypeptide.

“Naturally occurring NMU1 polypeptide” refers to a polypeptide producedby cells which have not been genetically engineered and specificallycontemplates various polypeptides arising from post-translationalmodifications of the polypeptide including but not limited toacetylation, carboxylation, glycosylation, phosphorylation, lipidationand acylation.

“Derivative” refers to polypeptides which have been chemically modifiedby techniques such as ubiquitination, labeling (see above), pegylation(derivatization with polyethylene glycol), and chemical insertion orsubstitution of amino acids such as ornithine which do not normallyoccur in human proteins.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine.

“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined byproducing the peptide synthetically while systematically makinginsertions, deletions, or substitutions of nucleotides in the sequenceusing recombinant DNA techniques.

A “signal sequence” or “leader sequence” can be used, when desired, todirect the polypeptide through a membrane of a cell. Such a sequence maybe naturally present on the polypeptides of the present invention orprovided from heterologous sources by recombinant DNA techniques.

An “oligopeptide” is a short stretch of amino acid residues and may beexpressed from an oligonucleotide. Oligopeptides comprise a stretch ofamino acid residues of at least 3, 5, 10 amino acids and at most 10, 15,25 amino acids, typically of at least 9 to 13 amino acids, and ofsufficient length to display biological and/or antigenic activity.

“Inhibitor” is any substance which retards or prevents a chemical orphysiological reaction or response. Common inhibitors include but arenot limited to antisense molecules, antibodies, and antagonists.

“Standard expression” is a quantitative or qualitative measurement forcomparison. It is based on a statistically appropriate number of normalsamples and is created to use as a basis of comparison when performingdiagnostic assays, running clinical trials, or following patienttreatment profiles.

“Animal” as used herein may be defined to include human, domestic (e.g.,cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) ortest species (e.g., mouse, rat, rabbit, etc.).

A “NMU1 polynucleotide”, within the meaning of the invention, shall beunderstood as being a nucleic acid molecule selected from a groupconsisting of

-   (i) nucleic acid molecules encoding a polypeptide comprising the    amino acid sequence of SEQ ID NO: 2,-   (ii) nucleic acid molecules comprising the sequence of SEQ ID NO: 1,-   (iii) nucleic acid molecules having the sequence of SEQ ID NO: 1,-   (iv) nucleic acid molecules the complementary strand of which    hybridizes under stringent conditions to a nucleic acid molecule of    (i), (ii), or (iii); and-   (v) nucleic acid molecules the sequence of which differs from the    sequence of a nucleic acid molecule of (iii) due to the degeneracy    of the genetic code;    wherein the polypeptide encoded by said nucleic acid molecule has    NMU1 activity.

A “NMU1 polypeptide”, within the meaning of the invention, shall beunderstood as being a polypeptide selected from a group consisting of

-   (i) polypeptides having the sequence of SEQ ID NO: 2,-   (ii) polypeptides comprising the sequence of SEQ ID NO: 2,-   (iii) polypeptides encoded by NMU1 polynucleotides; and-   (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80%    homology with a polypeptide of (i), (ii), or (iii);    wherein said polypeptide has NMU1 activity.

The nucleotide sequences encoding a NMU1 (or their complement) havenumerous applications in techniques known to those skilled in the art ofmolecular biology. These techniques include use as hybridization probes,use in the construction of oligomers for PCR, use for chromosome andgene mapping, use in the recombinant production of NMU1, and use ingeneration of antisense DNA or RNA, their chemical analogs and the like.Uses of nucleotides encoding a NMU1 disclosed herein are exemplary ofknown techniques and are not intended to limit their use in anytechnique known to a person of ordinary skill in the art. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown, e.g., the triplet genetic code, specific base pair interactions,etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of NMU1-encodingnucleotide sequences may be produced. Some of these will only bearminimal homology to the nucleotide sequence of the known and naturallyoccurring NMU1. The invention has specifically contemplated each andevery possible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequence of naturally occurring NMU1,and all such variations are to be considered as being specificallydisclosed.

Although the nucleotide sequences which encode a NMU1, its derivativesor its variants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring NMU1 polynucleotide under stringentconditions, it may be advantageous to produce nucleotide sequencesencoding NMU1 polypeptides or its derivatives possessing a substantiallydifferent codon usage. Codons can be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding a NMU1polypeptide and/or its derivatives without altering the encoded aminoacid sequence include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

Nucleotide sequences encoding a NMU1 polypeptide may be joined to avariety of other nucleotide sequences by means of well establishedrecombinant DNA techniques. Useful nucleotide sequences for joining toNMU1 polynucleotides include an assortment of cloning vectors such asplasmids, cosmids, lambda phage derivatives, phagemids, and the like.Vectors of interest include expression vectors, replication vectors,probe generation vectors, sequencing vectors, etc. In general, vectorsof interest may contain an origin of replication functional in at leastone organism, convenient restriction endonuclease sensitive sites, andselectable markers for one or more host cell systems.

Another aspect of the subject invention is to provide for NMU1-specifichybridization probes capable of hybridizing with naturally occurringnucleotide sequences encoding NMU1. Such probes may also be used for thedetection of similar GPCR encoding sequences and should preferably showat least 40% nucleotide identity to NMU1 polynucleotides. Thehybridization probes of the subject invention may be derived from thenucleotide sequence presented as SEQ ID NO: 1 or from genomic sequencesincluding promoter, enhancers or introns of the native gene.Hybridization probes may be labeled by a variety of reporter moleculesusing techniques well known in the art.

It will be recognized that many deletional or mutational analogs of NMU1polynucleotides will be effective hybridization probes for NMU1polynucleotides. Accordingly, the invention relates to nucleic acidsequences that hybridize with such NMU1 encoding nucleic acid sequencesunder stringent conditions.

“Stringent conditions” refers to conditions that allow for thehybridization of substantially related nucleic acid sequences. Forinstance, such conditions will generally allow hybridization of sequencewith at least about 85% sequence identity, preferably with at leastabout 90% sequence identity, more preferably with at least about 95%sequence identity. Hybridization conditions and probes can be adjustedin well-characterized ways to achieve selective hybridization ofhuman-derived probes. Stringent conditions, within the meaning of theinvention are 65° C. in a buffer containing 1 mM EDTA, 0.5 M NaHPO₄ (pH7.2), 7% (w/v) SDS.

Nucleic acid molecules that will hybridize to NMU1 polynucleotides understringent conditions can be identified functionally. Without limitation,examples of the uses for hybridization probes include: histochemicaluses such as identifying tissues that express NMU1; measuring mRNAlevels, for instance to identify a sample's tissue type or to identifycells that express abnormal levels of NMU1; and detecting polymorphismsof NMU1.

PCR provides additional uses for oligonucleotides based upon thenucleotide sequence which encodes NMU1. Such probes used in PCR may beof recombinant origin, chemically synthesized, or a mixture of both.Oligomers may comprise discrete nucleotide sequences employed underoptimized conditions for identification of NMU1 in specific tissues ordiagnostic use. The same two oligomers, a nested set of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for identification of closely related DNAs or RNAs.

Rules for designing polymerase chain reaction (“PCR”) primers are nowestablished, as reviewed by PCR Protocols. Degenerate primers, i.e.,preparations of primers that are heterogeneous at given sequencelocations, can be designed to amplify nucleic acid sequences that arehighly homologous to, but not identical with NMU1. Strategies are nowavailable that allow for only one of the primers to be required tospecifically hybridize with a known sequence. For example, appropriatenucleic acid primers can be ligated to the nucleic acid sought to beamplified to provide the hybridization partner for one of the primers.In this way, only one of the primers need be based on the sequence ofthe nucleic acid sought to be amplified.

PCR methods for amplifying nucleic acid will utilize at least twoprimers. One of these primers will be capable of hybridizing to a firststrand of the nucleic acid to be amplified and of priming enzyme-drivennucleic acid synthesis in a first direction. The other will be capableof hybridizing the reciprocal sequence of the first strand (if thesequence to be amplified is single stranded, this sequence willinitially be hypothetical, but will be synthesized in the firstamplification cycle) and of priming nucleic acid synthesis from thatstrand in the direction opposite the first direction and towards thesite of hybridization for the first primer. Conditions for conductingsuch amplifications, particularly under preferred stringenthybridization conditions, are well known.

Other means of producing specific hybridization probes for NMU1 includethe cloning of nucleic acid sequences encoding NMU1 or NMU1 derivativesinto vectors for the production of mRNA probes. Such vectors are knownin the art, are commercially available and may be used to synthesize RNAprobes in vitro by means of the addition of the appropriate RNApolymerase as T7 or SP6 RNA polymerase and the appropriate reportermolecules.

It is possible to produce a DNA sequence, or portions thereof, entirelyby synthetic chemistry. After synthesis, the nucleic acid sequence canbe inserted into any of the many available DNA vectors and theirrespective host cells using techniques which are well known in the art.Moreover, synthetic chemistry may be used to introduce mutations intothe nucleotide sequence. Alternately, a portion of sequence in which amutation is desired can be synthesized and recombined with longerportion of an existing genomic or recombinant sequence.

NMU1 polynucleotides may be used to produce a purified oligo- orpolypeptide using well known methods of recombinant DNA technology. Theoligopeptide may be expressed in a variety of host cells, eitherprokaryotic or eukaryotic. Host cells may be from the same species fromwhich the nucleotide sequence was derived or from a different species.Advantages of producing an oligonucleotide by recombinant DNA technologyinclude obtaining adequate amounts of the protein for purification andthe availability of simplified purification procedures.

Quantitative Determinations of Nucleic Acids

An important step in the molecular genetic analysis of human disease isoften the enumeration of the copy number of a nucleis acid or therelative expression of a gene in particular tissues.

Several different approaches are currently available to makequantitative determinations of nucleic acids. Chromosome-basedtechniques, such as comparative genomic hybridization (CGH) andfluorescent in situ hybridization (FISH) facilitate efforts tocytogenetically localize genomic regions that are altered in tumorcells.

Regions of genomic alteration can be narrowed further using loss ofheterozygosity analysis (LOH), in which disease DNA is analyzed andcompared with normal DNA for the loss of a heterozygous polymorphicmarker. The first experiments used restriction fragment lengthpolymorphisms (RFLPs) [Johnson, (1989)], or hypervariable minisatelliteDNA [Barnes, 2000]. In recent years LOH has been performed primarilyusing PCR amplification of microsatellite markers and electrophoresis ofthe radiolabeled [Jeffreys, (1985)] or fluorescently labeled PCRproducts [Weber, (1990)] and compared between paired normal and diseaseDNAs.

A number of other methods have also been developed to quantify nucleicacids [Gergen, [1992]. More recently, PCR and RT-PCR methods have beendeveloped which are capable of measuring the amount of a nucleic acid ina sample. One approach, for example, measures PCR product quantity inthe log phase of the reaction before the formation of reaction productsplateaus [Thomas, (1980)].

A gene sequence contained in all samples at relatively constant quantityis typically utilized for sample amplification efficiency normalization.This approach, however, suffers from several drawbacks. The methodrequires that each sample has equal input amounts of the nucleic acidand that the amplification efficiency between samples is identical untilthe time of analysis. Furthermore, it is difficult using theconventional methods of PCR quantitation such as gel electrophoresis orplate capture hybridization to determine that all samples are in factanalyzed during the log phase of the reaction as required by the method.

Another method called quantitative competitive (QC)-PCR, as the nameimplies, relies on the inclusion of an internal control competitor ineach reaction [Piatak, (1993), BioTechniques]. The efficiency of eachreaction is normalized to the internal competitor. A known amount ofinternal competitor is typically added to each sample. The unknowntarget PCR product is compared with the known competitor PCR product toobtain relative quantitation. A difficulty with this general approachlies in developing an internal control that amplifies with the sameefficiency than the target molecule.

5′ Fluorogenic Nuclease Assays

Fluorogenic nuclease assays are a real time quantitation method thatuses a probe to monitor formation of amplification product. The basisfor this method of monitoring the formation of amplification product isto measure continuously PCR product accumulation using a dual-labeledfluorogenic oligonucleotide probe, an approach frequently referred to inthe literature simply as the “TaqMan method” [Piatak, (1993), Science;Heid, (1996); Gibson, (1996); Holland. (1991)].

The probe used in such assays is typically a short (about 20-25 bases)oligonucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is attached to a reporter dye and the 3′terminus is attached to a quenching dye, although the dyes could beattached at other locations on the probe as well. The probe is designedto have at least substantial sequence complementarity with the probebinding site. Upstream and downstream PCR primers which bind to flankingregions of the locus are added to the reaction mixture. When the probeis intact, energy transfer between the two fluorophors occurs and thequencher quenches emission from the reporter. During the extension phaseof PCR, the probe is cleaved by the 5′ nuclease activity of a nucleicacid polymerase such as Taq polymerase, thereby releasing the reporterfrom the oligonucleotide-quencher and resulting in an increase ofreporter emission intensity which can be measured by an appropriatedetector.

One detector which is specifically adapted for measuring fluorescenceemissions such as those created during a fluorogenic assay is the ABI7700 or 4700 HT manufactured by Applied Biosystems, Inc. in Foster City,Calif. The ABI 7700 uses fiber optics connected with each well in a 96-or 384 well PCR tube arrangement. The instrument includes a laser forexciting the labels and is capable of measuring the fluorescence spectraintensity from each tube with continuous monitoring during PCRamplification. Each tube is reexamined every 8.5 seconds.

Computer software provided with the instrument is capable of recordingthe fluorescence intensity of reporter and quencher over the course ofthe amplification. The recorded values will then be used to calculatethe increase in normalized reporter emission intensity on a continuousbasis. The increase in emission intensity is plotted versus time, i.e.,the number of amplification cycles, to produce a continuous measure ofamplification. To quantify the locus in each amplification reaction, theamplification plot is examined at a point during the log phase ofproduct accumulation. This is accomplished by assigning a fluorescencethreshold intensity above background and determining the point at whicheach amplification plot crosses the threshold (defined as the thresholdcycle number or Ct). Differences in threshold cycle number are used toquantify the relative amount of PCR target contained within each tube.Assuming that each reaction functions at 100% PCR efficiency, adifference of one Ct represents a two-fold difference in the amount ofstarting template. The fluorescence value can be used in conjunctionwith a standard curve to determine the amount of amplification productpresent.

Non-Probe-Based Detection Methods

A variety of options are available for measuring the amplificationproducts as they are formed. One method utilizes labels, such as dyes,which only bind to double stranded DNA. In this type of approach,amplification product (which is double stranded) binds dye molecules insolution to form a complex. With the appropriate dyes, it is possible todistinguish between dye molecules free in solution and dye moleculesbound to amplification product. For example, certain dyes fluoresce onlywhen bound to amplification product. Examples of dyes which can be usedin methods of this general type include, but are not limited to, SyberGreen.™. and Pico Green from Molecular Probes, Inc. of Eugene, Oreg.,ethidium bromide, propidium iodide, chromomycin, acridine orange,Hoechst 33258, Toto-1, Yoyo-1, DAPI (4′,6-diamidino-2-phenylindolehydrochloride).

Another real time detection technique measures alteration in energyfluorescence energy transfer between fluorophors conjugated with PCRprimers [Livak, (1995)].

Probe-Based Detection Methods

These detection methods involve some alteration to the structure orconformation of a probe hybridized to the locus between theamplification primer pair. In some instances, the alteration is causedby the template-dependent extension catalyzed by a nucleic acidpolymerase during the amplification process. The alteration generates adetectable signal which is an indirect measure of the amount ofamplification product formed.

For example, some methods involve the degradation or digestion of theprobe during the extension reaction. These methods are a consequence ofthe 5′-3′ nuclease activity associated with some nucleic acidpolymerases. Polymerases having this activity cleave mononucleotides orsmall oligonucleotides from an oligonucleotide probe annealed to itscomplementary sequence located within the locus.

The 3′ end of the upstream primer provides the initial binding site forthe nucleic acid polymerase. As the polymerase catalyzes extension ofthe upstream primer and encounters the bound probe, the nucleic acidpolymerase displaces a portion of the 5′ end of the probe and throughits nuclease activity cleaves mononucleotides or oligonucleotides fromthe probe.

The upstream primer and the probe can be designed such that they annealto the complementary strand in close proximity to one another. In fact,the 3′ end of the upstream primer and the 5′ end of the probe may abutone another. In this situation, extension of the upstream primer is notnecessary in order for the nucleic acid polymerase to begin cleaving theprobe. In the case in which intervening nucleotides separate theupstream primer and the probe, extension of the primer is necessarybefore the nucleic acid polymerase encounters the 5′ end of the probe.Once contact occurs and polymerization continues, the 5′-3′ exonucleaseactivity of the nucleic acid polymerase begins cleaving mononucleotidesor oligonucleotides from the 5′ end of the probe. Digestion of the probecontinues until the remaining portion of the probe dissociates from thecomplementary strand.

In solution, the two end sections can hybridize with each other to forma hairpin loop. In this conformation, the reporter and quencher dye arein sufficiently close proximity that fluorescence from the reporter dyeis effectively quenched by the quencher dye. Hybridized probe, incontrast, results in a linearized conformation in which the extent ofquenching is decreased. Thus, by monitoring emission changes for the twodyes, it is possible to indirectly monitor the formation ofamplification product.

Probes

The labeled probe is selected so that its sequence is substantiallycomplementary to a segment of the test locus or a reference locus. Asindicated above, the nucleic acid site to which the probe binds shouldbe located between the primer binding sites for the upstream anddownstream amplification primers.

Primers

The primers used in the amplification are selected so as to be capableof hybridizing to sequences at flanking regions of the locus beingamplified. The primers are chosen to have at least substantialcomplementarity with the different strands of the nucleic acid beingamplified. When a probe is utilized to detect the formation ofamplification products, the primers are selected in such that they flankthe probe, i.e. are located upstream and downstream of the probe.

The primer must have sufficient length so that it is capable of primingthe synthesis of extension products in the presence of an agent forpolymerization. The length and composition of the primer depends on manyparameters, including, for example, the temperature at which theannealing reaction is conducted, proximity of the probe binding site tothat of the primer, relative concentrations of the primer and probe andthe particular nucleic acid composition of the probe. Typically theprimer includes 15-30 nucleotides. However, the length of the primer maybe more or less depending on the complexity of the primer binding siteand the factors listed above.

Labels for Probes and Primers

The labels used for labeling the probes or primers of the currentinvention and which can provide the signal corresponding to the quantityof amplification product can take a variety of forms. As indicated abovewith regard to the 5′ fluorogenic nuclease method, a fluorescent signalis one signal which can be measured. However, measurements may also bemade, for example, by monitoring radioactivity, colorimetry, absorption,magnetic parameters, or enzymatic activity. Thus, labels which can beemployed include, but are not limited to, fluorophors, chromophores,radioactive isotopes, electron dense reagents, enzymes, and ligandshaving specific binding partners (e.g., biotin-avidin).

Monitoring changes in fluorescence is a particularly useful way tomonitor the accumulation of amplification products. A number of labelsuseful for attachment to probes or primers are commercially availableincluding fluorescein and various fluorescein derivatives such as FAM,HEX, TET and JOE (all which are available from Applied Biosystems,Foster City, Calif.); lucifer yellow, and coumarin derivatives.

Labels may be attached to the probe or primer using a variety oftechniques and can be attached at the 5′ end, and/or the 3′ end and/orat an internal nucleotide. The label can also be attached to spacer armsof various sizes which are attached to the probe or primer. These spacerarms are useful for obtaining a desired distance between multiple labelsattached to the probe or primer.

In some instances, a single label may be utilized; whereas, in otherinstances, such as with the 5′ fluorogenic nuclease assays for example,two or more labels are attached to the probe. In cases wherein the probeincludes multiple labels, it is generally advisable to maintain spacingbetween the labels which is sufficient to permit separation of thelabels during digestion of the probe through the 5′-3′ nuclease activityof the nucleic acid polymerase.

Patients Exhibiting Symptoms of Disease

A number of diseases are associated with changes in the copy number of acertain gene. For patients having symptoms of a disease, the real-timePCR method can be used to determine if the patient has copy numberalterations which are known to be linked with diseases that areassociated with the symptoms the patient has.

NMU1 Expression

NMU1 Fusion Proteins

Fusion proteins are useful for generating antibodies against NMU1polypeptides and for use in various assay systems. For example, fusionproteins can be used to identify proteins which interact with portionsof NMU1 polypeptides. Protein affinity chromatography or library-basedassays for protein-protein interactions, such as the yeast two-hybrid orphage display systems, can be used for this purpose. Such methods arewell known in the art and also can be used as drug screens.

A NMU1 fusion protein comprises two polypeptide segments fused togetherby means of a peptide bond. The first polypeptide segment can compriseat least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, or 275contiguous amino acids of SEQ ID NO: 2 or of a biologically activevariant, such as those described above. The first polypeptide segmentalso can comprise full-length NMU1.

The second polypeptide segment can be a full-length protein or a proteinfragment. Proteins commonly used in fusion protein construction include,but are not limited to β galactosidase, β-glucuronidase, greenfluorescent protein (GFP), autofluorescent proteins, including bluefluorescent protein (BFP), glutathione-S-transferase (GST), luciferase,horseradish peroxidase (HRP), and chloramphenicol acetyltransferase(CAT). Additionally, epitope tags are used in fusion proteinconstructions, including histidine (His) tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSVG tags, and thioredoxin (Trx)tags. Other fusion constructions can include maltose binding protein(MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA bindingdomain fusions, herpes simplex virus (HSV) BP16 protein fusions andG-protein fusions (for example G(alpha)16, Gs, Gi). A fusion proteinalso can be engineered to contain a cleavage site located adjacent tothe NMU1.

Preparation of Polynucleotides

A naturally occurring NMU1 polynucleotide can be isolated free of othercellular components such as membrane components, proteins, and lipids.Polynucleotides can be made by a cell and isolated using standardnucleic acid purification techniques, or synthesized using anamplification technique, such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide can be used to obtain isolated NMU1 polynucleotides. Forexample, restriction enzymes and probes can be used to isolatepolynucleotide fragments which comprises NMU1 nucleotide sequences.Isolated polynucleotides are in preparations which are free or at least70, 80, or 90% free of other molecules.

NMU1 cDNA molecules can be made with standard molecular biologytechniques, using NMU1 mRNA as a template. NMU1 cDNA molecules canthereafter be replicated using molecular biology techniques known in theart. An amplification technique, such as PCR, can be used to obtainadditional copies of polynucleotides of the invention, using eitherhuman genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesizesNMU1 polynucleotides. The degeneracy of the genetic code allowsalternate nucleotide sequences to be synthesized which will encode NMU1having, for example, an amino acid sequence shown in SEQ ID NO: 2 or abiologically active variant thereof.

Extending Polynucleotides

Various PCR-based methods can be used to extend nucleic acid sequencesencoding human NMU1, for example to detect upstream sequences of NMU1gene such as promoters and regulatory elements. For example,restriction-site PCR uses universal primers to retrieve unknown sequenceadjacent to a known locus. Genomic DNA is first amplified in thepresence of a primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR also can be used to amplify or extend sequences usingdivergent primers based on a known region. Primers can be designed usingcommercially available software, such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68-72° C. The methoduses several restriction enzymes to generate a suitable fragment in theknown region of a gene. The fragment is then circularized byintramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. In this method, multiple restrictionenzyme digestions and ligations also can be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Randomly-primedlibraries are preferable, in that they will contain more sequences whichcontain the 5′ regions of genes. Use of a randomly primed library may beespecially preferable for situations in which an oligo d(T) library doesnot yield a full-length cDNA. Genomic libraries can be useful forextension of sequence into 5′ non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used toanalyze the size or confirm the nucleotide sequence of PCR or sequencingproducts. For example, capillary sequencing can employ flowable polymersfor electrophoretic separation, four different fluorescent dyes (one foreach nucleotide) which are laser activated, and detection of the emittedwavelengths by a charge coupled device camera. Output/light intensitycan be converted to electrical signal using appropriate equipment andsoftware (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and theentire process from loading of samples to computer analysis andelectronic data display can be computer controlled. Capillaryelectrophoresis is especially preferable for the sequencing of smallpieces of DNA which might be present in limited amounts in a particularsample.

Obtaining Polypeptides

NMU1 can be obtained, for example, by purification from human cells, byexpression of NMU1 polynucleotides, or by direct chemical synthesis.

Protein Purification

NMU1 can be purified from any human cell which expresses the receptor,including those which have been transfected with expression constructswhich express NMU1. A purified NMU1 is separated from other compoundswhich normally associate with NMU1 in the cell, such as certainproteins, carbohydrates, or lipids, using methods well-known in the art.Such methods include, but are not limited to, size exclusionchromatography, ammonium sulfate fractionation, ion exchangechromatography, affinity chromatography, and preparative gelelectrophoresis.

Expression of NMU1 Polynucleotides

To express NMU1, NMU1 polynucleotides can be inserted into an expressionvector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing sequences encoding NMU1 and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination.

A variety of expression vector/host systems can be utilized to containand express sequences encoding NMU1. These include, but are not limitedto, microorganisms, such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors, insect cell systems infectedwith virus expression vectors (e.g., baculovirus), plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translatedregions of the vector—enhancers, promoters, 5′ and 3′ untranslatedregions—which interact with host cellular proteins to carry outtranscription and translation. Such elements can vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, can be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.)or pSPORT1 plasmid (Life Technologies) and the like can be used. Thebaculovirus polyhedrin promoter can be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) can be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferable. If it is necessary to generate a cell line thatcontains multiple copies of a nucleotide sequence encoding NMU1, vectorsbased on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected.For example, when a large quantity of NMU1 is needed for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified can be used. Such vectors include,but are not limited to, multifunctional E. coli cloning and expressionvectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, asequence encoding NMU1 can be ligated into the vector in frame withsequences for the amino-terminal Met and the subsequent 7 residues ofβ-galactosidase so that a hybrid protein is produced. pIN vectors orpGEX vectors (Promega, Madison, Wis.) also can be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption to glutathione-agarose beadsfollowed by elution in the presence of free glutathione. Proteins madein such systems can be designed to include heparin, thrombin, or factorXa protease cleavage sites so that the cloned polypeptide of interestcan be released from the GST moiety at will.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequencesencoding NMU1 can be driven by any of a number of promoters. Forexample, viral promoters such as the 35S and 19S promoters of CaMV canbe used alone or in combination with the omega leader sequence from TMV.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters can be used. These constructs can be introducedinto plant cells by direct DNA transformation or by pathogen-mediatedtransfection.

An insect system also can be used to express NMU1. For example, in onesuch system Autographa californica nuclear polyhedrosis virus (AcNPV) isused as a vector to express foreign genes in Spodoptera frugiperda cellsor in Trichoplusia larvae. Sequences encoding NMU1 can be cloned into anon-essential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofNMU1 will render the polyhedrin gene inactive and produce recombinantvirus lacking coat protein. The recombinant viruses can then be used toinfect S. frugiperda cells or Trichoplusia larvae in which NMU1 can beexpressed.

Mammalian Expression Systems

A number of viral-based expression systems can be used to express NMU1in mammalian host cells. For example, if an adenovirus is used as anexpression vector, sequences encoding NMU1 can be ligated into anadenovirus transcription/translation complex comprising the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome can be used to obtain a viable viruswhich is capable of expressing NMU1 in infected host cells [Engelhard,1994)]. If desired, transcription enhancers, such as the Rous sarcomavirus (RSV) enhancer, can be used to increase expression in mammalianhost cells.

Human artificial chromosomes (HACs) also can be used to deliver largerfragments of DNA than can be contained and expressed in a plasmid. HACsof 6M to 10M are constructed and delivered to cells via conventionaldelivery methods (e.g., liposomes, polycationic amino polymers, orvesicles). Specific initiation signals also can be used to achieve moreefficient translation of sequences encoding NMU1. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding NMU1, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals (including the ATG initiationcodon) should be provided. The initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons can be of various origins,both natural and synthetic.

Host Cells

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed NMU1 inthe desired fashion. Such modifications of the polypeptide include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation, and acylation. Post-translationalprocessing which cleaves a “prepro” form of the polypeptide also can beused to facilitate correct insertion, folding and/or function. Differenthost cells which have specific cellular machinery and characteristicmechanisms for post-translational activities (e.g., CHO, HeLa, MDCK,HEK293, and WI38), are available from the American Type CultureCollection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209)and can be chosen to ensure the correct modification and processing ofthe foreign protein.

Stable expression is preferred for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably express NMU1can be transformed using expression vectors which can contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells can be allowed to grow for 1-2days in an enriched medium before they are switched to a selectivemedium. The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced NMU1 sequences. Resistant clones ofstably transformed cells can be proliferated using tissue culturetechniques appropriate to the cell type. Any number of selection systemscan be used to recover transformed cell lines. These include, but arenot limited to, the herpes simplex virus thymidine kinase [Logan,(1984)] and adenine phosphoribosyltransferase [Wigler, (1977)] geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate [Lowy, (1980)], npt confers resistance to theaminoglycosides, neomycin and G418 [Wigler, (1980)], and als and patconfer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively [Colbere-Garapin, 1981]. Additionalselectable genes have been described. For example, trpB allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine [Murray, (1992)]. Visible markerssuch as anthocyanins, β-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, can be used to identifytransformants and to quantify the amount of transient or stable proteinexpression attributable to a specific vector system.

Detecting Polypeptide Expression

Although the presence of marker gene expression suggests that a NMU1polynucleotide is also present, its presence and expression may need tobe confirmed. For example, if a sequence encoding NMU1 is insertedwithin a marker gene sequence, transformed cells containing sequenceswhich encode NMU1 can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding NMU1 under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of NMU1 polynucleotide.

Alternatively, host cells which contain a NMU1 polynucleotide and whichexpress NMU1 can be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassaytechniques which include membrane, solution, or chip-based technologiesfor the detection and/or quantification of nucleic acid or protein. Forexample, the presence of a polynucleotide sequence encoding NMU1 can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding NMU1.Nucleic acid amplification-based assays involve the use ofoligonucleotides selected from sequences encoding NMU1 to detecttransformants which contain a NMU1 polynucleotide.

A variety of protocols for detecting and measuring the expression ofNMU1, using either polyclonal or monoclonal antibodies specific for thepolypeptide, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayusing monoclonal antibodies reactive to two non-interfering epitopes onNMU1 can be used, or a competitive binding assay can be employed.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding NMU1 includeoligolabeling, nick translation, endlabeling, or PCR amplification usinga labeled nucleotide. Alternatively, sequences encoding NMU1 can becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and can be used tosynthesize RNA probes in vitro by addition of labeled nucleotides and anappropriate RNA polymerase such as T7, T3, or SP6. These procedures canbe conducted using a variety of commercially available kits (AmershamPharmacia Biotech, Promega, and US Biochemical). Suitable reportermolecules or labels which can be used for ease of detection includeradionuclides, enzymes, and fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with NMU1 polynucleotides can be cultured underconditions suitable for the expression and recovery of the protein fromcell culture. The polypeptide produced by a transformed cell can besecreted or contained intracellularly depending on the sequence and/orthe vector used. As will be understood by those of skill in the art,expression vectors containing NMU1 polynucleotides can be designed tocontain signal sequences which direct secretion of soluble NMU1 througha prokaryotic or eukaryotic cell membrane or which direct the membraneinsertion of membrane-bound NMU1.

As discussed above, other constructions can be used to join a sequenceencoding NMU1 to a nucleotide sequence encoding a polypeptide domainwhich will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). Inclusion of cleavable linker sequences such as thosespecific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.)between the purification domain and NMU1 also can be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing NMU1 and 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification by IMAC (immobilized metal ion affinitychromatography) Maddox, (1983)], while the enterokinase cleavage siteprovides a means for purifying NMU1 from the fusion protein [Porath,(1992)].

Chemical Synthesis

Sequences encoding NMU1 can be synthesized, in whole or in part, usingchemical methods well known in the art. Alternatively, NMU1 itself canbe produced using chemical methods to synthesize its amino acidsequence, such as by direct peptide synthesis using solid-phasetechniques. Protein synthesis can either be performed using manualtechniques or by automation. Automated synthesis can be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Optionally, fragments of NMU1 can be separately synthesized andcombined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography. The composition of asynthetic NMU1 can be confirmed by amino acid analysis or sequencing.Additionally, any portion of the amino acid sequence of NMU1 can bealtered during direct synthesis and/or combined using chemical methodswith sequences from other proteins to produce a variant polypeptide or afusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may beadvantageous to produce NMU1 polynucleotides possessing non-naturallyoccurring codons. For example, codons preferred by a particularprokaryotic or eukaryotic host can be selected to increase the rate ofprotein expression or to produce an RNA transcript having desirableproperties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences referred to herein can be engineered usingmethods generally known in the art to alter NMU1 polynucleotides for avariety of reasons, including but not limited to, alterations whichmodify the cloning, processing, and/or expression of the polypeptide ormRNA product. DNA shuffling by random fragmentation and PCR reassemblyof gene fragments and synthetic oligonucleotides can be used to engineerthe nucleotide sequences. For example, site-directed mutagenesis can beused to insert new restriction sites, alter glycosylation patterns,change codon preference, produce splice variants, introduce mutations,and so forth.

Antibodies

Any type of antibody known in the art can be generated to bindspecifically to an epitope of NMU1.

“Antibody” as used herein includes intact immunoglobulin molecules, aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding an epitope of NMU1. Typically, at least 6, 8, 10, or12 contiguous amino acids are required to form an epitope. However,epitopes which involve non-contiguous amino acids may require more,e.g., at least 15, 25, or 50 amino acid. An antibody which specificallybinds to an epitope of NMU1 can be used therapeutically, as well as inimmunochemical assays, such as Western blots, ELISAs, radioimmunoassays,immunohistochemical assays, immunoprecipitations, or otherimmunochemical assays known in the art. Various immunoassays can be usedto identify antibodies having the desired specificity. Numerousprotocols for competitive binding or immunoradiometric assays are wellknown in the art. Such immunoassays typically involve the measurement ofcomplex formation between an immunogen and an antibody whichspecifically binds to the NMU1 immunogen.

Typically, an antibody which specifically binds to NMU1 provides adetection signal at least 5-, 10-, or 20-fold higher than a detectionsignal provided with other proteins when used in an immunochemicalassay. Preferably, antibodies which specifically bind to NMU1 do notdetect other proteins in immunochemical assays and can immunoprecipitateNMU1 from solution.

NMU1 can be used to immunize a mammal, such as a mouse, rat, rabbit,guinea pig, monkey, or human, to produce polyclonal antibodies. Ifdesired, NMU1 can be conjugated to a carrier protein, such as bovineserum albumin, thyroglobulin, and keyhole limpet hemocyanin. Dependingon the host species, various adjuvants can be used to increase theimmunological response. Such adjuvants include, but are not limited to,Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surfaceactive substances (e.g., lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to NMU1 can be preparedusing any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These techniques include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique [Roberge, (1995)].

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used. Monoclonal and other antibodies alsocan be “humanized” to prevent a patient from mounting an immune responseagainst the antibody when it is used therapeutically. Such antibodiesmay be sufficiently similar in sequence to human antibodies to be useddirectly in therapy or may require alteration of a few key residues.Sequence differences between rodent antibodies and human sequences canbe minimized by replacing residues which differ from those in the humansequences by site directed mutagenesis of individual residues or bygrating of entire complementarity determining regions. Antibodies whichspecifically bind to NMU1 can contain antigen binding sites which areeither partially or fully humanized, as disclosed in U.S. Pat. No.5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to NMU1. Antibodies withrelated specificity, but of distinct idiotypic composition, can begenerated by chain shuffling from random combinatorial immunoglobinlibraries. Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template.Single-chain antibodies can be mono- or bispecific, and can be bivalentor tetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught. A nucleotide sequence encoding a single-chainantibody can be constructed using manual or automated nucleotidesynthesis, cloned into an expression construct using standardrecombinant DNA methods, and introduced into a cell to express thecoding sequence, as described below. Alternatively, single-chainantibodies can be produced directly using, for example, filamentousphage technology.

Antibodies which specifically bind to NMU1 also can be produced byinducing in vivo production in the lymphocyte population or by screeningimmunoglobulin libraries or panels of highly specific binding reagents.Other types of antibodies can be constructed and used therapeutically inmethods of the invention. For example, chimeric antibodies can beconstructed as disclosed in WO 93/03151. Binding proteins which arederived from immunoglobulins and which are multivalent andmultispecific, such as the “diabodies” described in WO 94/13804, alsocan be prepared.

Antibodies according to the invention can be purified by methods wellknown in the art. For example, antibodies can be affinity purified bypassage over a column to which NMU1 is bound. The bound antibodies canthen be eluted from the column using a buffer with a high saltconcentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which arecomplementary to a specific DNA or RNA sequence. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form complexes and block either transcription ortranslation. Preferably, an antisense oligonucleotide is at least 11nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,45, or 50 or more nucleotides long. Longer sequences also can be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into a cell as described above to decrease the level ofNMU1 gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides,or a combination of both. Oligonucleotides can be synthesized manuallyor by an automated synthesizer, by covalently linking the 5′ end of onenucleotide with the 3′ end of another nucleotide with non-phosphodiesterinternucleotide linkages such alkylphosphonates, phosphorothioates,phosphorodithioates, alkylphosphonothioates, alkylphosphonates,phosphoramidates, phosphate esters, carbamates, acetamidate,carboxymethyl esters, carbonates, and phosphate triesters.

Modifications of NMU1 gene expression can be obtained by designingantisense oligonucleotides which will form duplexes to the control, 5′,or regulatory regions of the NMU1 gene. Oligonucleotides derived fromthe transcription initiation site, e.g., between positions −10 and +10from the start site, are preferred. Similarly, inhibition can beachieved using “triple helix” base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or chaperons. Therapeutic advances using triplexDNA have been described in the literature [Nicholls, (1993)]. Anantisense oligonucleotide also can be designed to block translation ofmRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formationbetween an antisense oligonucleotide and the complementary sequence of aNMU1 polynucleotide. Antisense oligonucleotides which comprise, forexample, 2, 3, 4, or 5 or more stretches of contiguous nucleotides whichare precisely complementary to a NMU1 polynucleotide, each separated bya stretch of contiguous nucleotides which are not complementary toadjacent NMU1 nucleotides, can provide sufficient targeting specificityfor NMU1 mRNA. Preferably, each stretch of complementary contiguousnucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.Noncomplementary intervening sequences are preferably 1, 2, 3, or 4nucleotides in length. One skilled in the art can easily use thecalculated melting point of an antisense-sense pair to determine thedegree of mismatching which will be tolerated between a particularantisense oligonucleotide and a particular NMU1 polynucleotide sequence.Antisense oligonucleotides can be modified without affecting theirability to hybridize to a NMU1 polynucleotide. These modifications canbe internal or at one or both ends of the antisense molecule. Forexample, internucleoside phosphate linkages can be modified by addingcholesteryl or diamine moieties with varying numbers of carbon residuesbetween the amino groups and terminal ribose. Modified bases and/orsugars, such as arabinose instead of ribose, or a 3′,5′-substitutedoligonucleotide in which the 3′ hydroxyl group or the 5′ phosphate groupare substituted, also can be employed in a modified antisenseoligonucleotide. These modified oligonucleotides can be prepared bymethods well known in the art.

Ribozymes

Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)].Ribozymes can be used to inhibit gene function by cleaving an RNAsequence, as is known in the art. The mechanism of ribozyme actioninvolves sequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Examplesinclude engineered hammerhead motif ribozyme molecules that canspecifically and efficiently catalyze endonucleolytic cleavage ofspecific nucleotide sequences. The coding sequence of a NMU1pol-nucleotide can be used to generate ribozymes which will specificallybind to mRNA transcribed from a NMU1 polynucleotide. Methods ofdesigning and constructing ribozymes which can cleave other RNAmolecules in trans in a highly sequence specific manner have beendeveloped and described in the art. For example, the cleavage activityof ribozymes can be targeted to specific RNAs by engineering a discrete“hybridization” region into the ribozyme. The hybridization regioncontains a sequence complementary to the target RNA and thusspecifically hybridizes with the Specific ribozyme cleavage sites withina NMU1 RNA target can be identified by scanning the target molecule forribozyme cleavage sites which include the following sequences: GUA, GUU,and GUC. Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target RNA containingthe cleavage site can be evaluated for secondary structural featureswhich may render the target inoperable. Suitability of candidate NMU1RNA targets also can be evaluated by testing accessibility tohybridization with complementary oligonucleotides using ribonucleaseprotection assays. The nucleotide sequences shown in SEQ ID NO: 1 andits complement provide sources of suitable hybridization regionsequences. Longer complementary sequences can be used to increase theaffinity of the hybridization sequence for the target. The hybridizingand cleavage regions of the ribozyme can be integrally related such thatupon hybridizing to the target RNA through the complementary regions,the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct.Mechanical methods, such as microinjection, liposome-mediatedtransfection, electroporation, or calcium phosphate precipitation, canbe used to introduce a ribozyme-containing DNA construct into cells inwhich it is desired to decrease NMU1 expression. Alternatively, if it isdesired that the cells stably retain the DNA construct, the constructcan be supplied on a plasmid and maintained as a separate element orintegrated into the genome of the cells, as is known in the art. Aribozyme-encoding DNA construct can include transcriptional regulatoryelements, such as a promoter element, an enhancer or UAS element, and atranscriptional terminator signal, for controlling transcription ofribozymes in the cells (U.S. Pat. No. 5,641,673). Ribozymes also can beengineered to provide an additional level of regulation, so thatdestruction of mRNA occurs only when both a ribozyme and a target geneare induced in the cells.

Screening/Screening Assays

Regulators

Regulators as used herein, refer to compounds that affect the activityof a NMU1 in vivo and/or in vivo. Regulators can be agonists andantagonists of a NMU1 polypeptide and can be compounds that exhert theireffect on the NMU1 activity via the expression, via post-translationalmodifications or by other means. Agonists of NMU1 are molecules which,when bound to NMU1, increase or prolong the activity of NMU1. Agonistsof NMU1 include proteins, nucleic acids, carbohydrates, small molecules,or any other molecule which activate NMU1. Antagonists of NMU1 aremolecules which, when bound to NMU1, decrease the amount or the durationof the activity of NMU1. Antagonists include proteins, nucleic acids,carbohydrates, antibodies, small molecules, or any other molecule whichdecrease the activity of NMU1.

The term “modulate,” as it appears herein, refers to a change in theactivity of NMU1 polypeptide. For example, modulation may cause anincrease or a decrease in protein activity, binding characteristics, orany other biological, functional, or immunological properties of NMU1.

As used herein, the terms “specific binding” or “specifically binding”refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein recognized by thebinding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope “A” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

The invention provides methods (also referred to herein as “screeningassays”) for identifying compounds which can be used for the treatmentof hematological and cardiovascular diseases, disorders of theperipheral and central nervous system, COPD, asthma, genito-urologicaldisorders and inflammation diseases. The methods entail theidentification of candidate or test compounds or agents (e.g., peptides,peptidomimetics, small molecules or other molecules) which bind to NMU1and/or have a stimulatory or inhibitory effect on the biologicalactivity of NMU1 or its expression and then determining which of thesecompounds have an effect on symtoms or diseases regarding thehematological and cardiovascular diseases, disorders of the peripheraland central nervous system, COPD, asthma, genito-urological disordersand inflammation diseases in an in vivo assay.

Candidate or test compounds or agents which bind to NMU1 and/or have astimulatory or inhibitory effect on the activity or the expression ofNMU1 are identified either in assays that employ cells which expressNMU1 on the cell surface (cell-based assays) or in assays with isolatedNMU1 (cell-free assays). The various assays can employ a variety ofvariants of NMU1 (e.g., full-length NMU1, a biologically active fragmentof NMU1, or a fusion protein which includes all or a portion of NMU1).Moreover, NMU1 can be derived from any suitable mammalian species (e.g.,human NMU1, rat NMU1 or murine NMU1). The assay can be a binding assayentailing direct or indirect measurement of the binding of a testcompound or a known NMU1 ligand to NMU1. The assay can also be anactivity assay entailing direct or indirect measurement of the activityof NMU1. The assay can also be an expression assay entailing direct orindirect measurement of the expression of NMU1 mRNA or NMU1 protein. Thevarious screening assays are combined with an in vivo assay entailingmeasuring the effect of the test compound on the symtoms ofhematological and cardiovascular diseases, disorders of the peripheraland central nervous system, COPD, asthma, genito-urological disordersand inflammation diseases.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of amembrane-bound (cell surface expressed) form of NMU1. Such assays canemploy full-length NMU1, a biologically active fragment of NMU1, or afusion protein which includes all or a portion of NMU1. As described ingreater detail below, the test compound can be obtained by any suitablemeans, e.g., from conventional compound libraries. Determining theability of the test compound to bind to a membrane-bound form of NMU1can be accomplished, for example, by coupling the test compound with aradioisotope or enzymatic label such that binding of the test compoundto the NMU1-expressing cell can be measured by detecting the labeledcompound in a complex. For example, the test compound can be labeledwith ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, the test compound can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In a competitive binding format, the assay comprises contacting NMU1expressing cell with a known compound which binds to NMU1 to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with the NMU1expressing cell, wherein determining the ability of the test compound tointeract with the NMU1 expressing cell comprises determining the abilityof the test compound to preferentially bind the NMU1 expressing cell ascompared to the known compound.

In another embodiment, the assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of NMU1 (e.g.,full-length NMU1, a biologically active fragment of NMU1, or a fusionprotein which includes all or a portion of NMU1) expressed on the cellsurface with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of themembrane-bound form of NMU1. Determining the ability of the testcompound to modulate the activity of the membrane-bound form of NMU1 canbe accomplished by any method suitable for measuring the activity ofNMU1, e.g., any method suitable for measuring the activity of aG-protein coupled receptor or other seven-transmembrane receptor(described in greater detail below). The activity of aseven-transmembrane receptor can be measured in a number of ways, notall of which are suitable for any given receptor. Among the measures ofactivity are: alteration in intracellular Ca²⁺ concentration, activationof phospholipase C, alteration in intracellular inositol triphosphate(IP3) concentration, alteration in intracellular diacylglycerol (DAG)concentration, and alteration in intracellular adenosine cyclic3′,5′-monophosphate (cAMP) concentration.

Determining the ability of the test compound to modulate the activity ofNMU1 can be accomplished, for example, by determining the ability ofNMU1 to bind to or interact with a target molecule. The target moleculecan be a molecule with which NMU1 binds or interacts with in nature, forexample, a molecule on the surface of a cell which expresses NMU1, amolecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. The target molecule can be acomponent of a signal transduction pathway which facilitatestransduction of an extracellular signal (e.g., a signal generated bybinding of a NMU1 ligand, through the cell membrane and into the cell.The target NMU1 molecule can be, for example, a second intracellularprotein which has catalytic activity or a protein which facilitates theassociation of downstream signaling molecules with NMU1.

Determining the ability of NMU1 to bind to or interact with a targetmolecule can be accomplished by one of the methods described above fordetermining direct binding. In one embodiment, determining the abilityof a polypeptide of the invention to bind to or interact with a targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (e.g., intracellular Ca²⁺, diacylglycerol, IP3, etc.), detectingcatalytic/enzymatic activity of the target on an appropriate substrate,detecting the induction of a reporter gene (e.g., a regulatory elementthat is responsive to a polypeptide of the invention operably linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a cellular response.

The present invention also includes cell-free assays. Such assaysinvolve contacting a form of NMU1 (e.g., full-length NMU1, abiologically active fragment of NMU1, or a fusion protein comprising allor a portion of NMU1) with a test compound and determining the abilityof the test compound to bind to NMU1. Binding of the test compound toNMU1 can be determined either directly or indirectly as described above.In one embodiment, the assay includes contacting NMU1 with a knowncompound which binds NMU1 to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with NMU1, wherein determining the ability of thetest compound to interact with NMU1 comprises determining the ability ofthe test compound to preferentially bind to NMU1 as compared to theknown compound.

The cell-free assays of the present invention are amenable to use ofeither a membrane-bound form of NMU1 or a soluble fragment thereof. Inthe case of cell-free assays comprising the membrane-bound form of thepolypeptide, it may be desirable to utilize a solubilizing agent suchthat the membrane-bound form of the polypeptide is maintained insolution. Examples of such solubilizing agents include but are notlimited to non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In various embodiments of the above assay methods of the presentinvention, it may be desirable to immobilize NMU1 (or a NMU1 targetmolecule) to facilitate separation of complexed from uncomplexed formsof one or both of the proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to NMU1, or interaction of NMU1with a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows one or both of theproteins to be bound to a matrix. For example, glutathione-S-transferase(GST) fusion proteins or glutathione-S-transferase fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or NMU1, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components andcomplex formation is measured either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of binding or activity ofNMU1 can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either NMU1 orits target molecule can be immobilized utilizing conjugation of biotinand streptavidin. Biotinylated polypeptide of the invention or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well known in the art (e.g., biotinylation kit, PierceChemicals; Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated plates (Pierce Chemical). Alternatively, antibodiesreactive with NMU1 or target molecules but which do not interfere withbinding of the polypeptide of the invention to its target molecule canbe derivatized to the wells of the plate, and unbound target orpolypeptide of the invention trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with NMU1 ortarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with NMU1 or target molecule.

The screening assay can also involve monitoring the expression of NMU1.For example, regulators of expression of NMU1 can be identified in amethod in which a cell is contacted with a candidate compound and theexpression of NMU1 protein or mRNA in the cell is determined. The levelof expression of NMU1 protein or mRNA the presence of the candidatecompound is compared to the level of expression of NMU1 protein or mRNAin the absence of the candidate compound. The candidate compound canthen be identified as a regulator of expression of NMU1 based on thiscomparison. For example, when expression of NMU1 protein or mRNA proteinis greater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of NMU1 protein or mRNA expression.Alternatively, when expression of NMU1 protein or mRNA is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of NMU1 protein or mRNA expression. The level of NMU1 proteinor mRNA expression in the cells can be determined by methods describedbelow.

Binding Assays

For binding assays, the test compound is preferably a small moleculewhich binds to and occupies the active site of NMU1 polypeptide, therebymaking the ligand binding site inaccessible to substrate such thatnormal biological activity is prevented. Examples of such smallmolecules include, but are not limited to, small peptides orpeptide-like molecules. Potential ligands which bind to a polypeptide ofthe invention include, but are not limited to, the natural ligands ofknown NMU1 GPCRs and analogues or derivatives thereof.

In binding assays, either the test compound or the NMU1 polypeptide cancomprise a detectable label, such as a fluorescent, radioisotopic,chemiluminescent, or enzymatic label, such as horseradish peroxidase,alkaline phosphatase, or luciferase. Detection of a test compound whichis bound to NMU1 polypeptide can then be accomplished, for example, bydirect counting of radioemmission, by scintillation counting, or bydetermining conversion of an appropriate substrate to a detectableproduct. Alternatively, binding of a test compound to a NMU1 polypeptidecan be determined without labeling either of the interactants. Forexample, a microphysiometer can be used to detect binding of a testcompound with a NMU1 polypeptide. A microphysiometer (e.g., Cytosensor™)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a test compound and NMU1 [Haseloff,(1988)].

Determining the ability of a test compound to bind to NMU1 also can beaccomplished using a technology such as real-time BimolecularInteraction Analysis (BIA) [McConnell, (1992); Sjolander, (1991)]. BIAis a technology for studying biospecific interactions in real time,without labeling any of the interactants (e.g., BIAcore™). Changes inthe optical phenomenon surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

In yet another aspect of the invention, a NMU1-like polypeptide can beused as a “bait protein” in a two-hybrid assay or three-hybrid assay[Szabo, (1995); U.S. Pat. No. 5,283,317), to identify other proteinswhich bind to or interact with NMU1 and modulate its activity.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct, polynucleotide encoding NMU1can be fused to a polynucleotide encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct a DNAsequence that encodes an unidentified protein (“prey” or “sample”) canbe fused to a polynucleotide that codes for the activation domain of theknown transcription factor. If the “bait” and the “prey” proteins areable to interact in vivo to form an protein-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ), which is operably linked to atranscriptional regulatory site responsive to the transcription factor.Expression of the reporter gene can be detected, and cell coloniescontaining the functional transcription factor can be isolated and usedto obtain the DNA sequence encoding the protein which interacts withNMU1.

It may be desirable to immobilize either the NMU1 (or polynucleotide) orthe test compound to facilitate separation of the bound form fromunbound forms of one or both of the interactants, as well as toaccommodate automation of the assay. Thus, either the NMU1-likepolypeptide (or polynucleotide) or the test compound can be bound to asolid support. Suitable solid supports include, but are not limited to,glass or plastic slides, tissue culture plates, microtiter wells, tubes,silicon chips, or particles such as beads (including, but not limitedto, latex, polystyrene, or glass beads). Any method known in the art canbe used to attach NMU1-like polypeptide (or polynucleotide) or testcompound to a solid support, including use of covalent and non-covalentlinkages, passive absorption, or pairs of binding moieties attachedrespectively to the polypeptide (or polynucleotide) or test compound andthe solid support. Test compounds are preferably bound to the solidsupport in an array, so that the location of individual test compoundscan be tracked. Binding of a test compound to NMU1 (or a polynucleotideencoding for NMU1) can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and microcentrifuge tubes.

In one embodiment, NMU1 is a fusion protein comprising a domain thatallows binding of NMU1 to a solid support. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and the non-adsorbed NMU1; themixture is then incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components. Binding of the interactants can be determined eitherdirectly or indirectly, as described above. Alternatively, the complexescan be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solidsupport also can be used in the screening assays of the invention. Forexample, either NMU1 (or a polynucleotide encoding NMU1) or a testcompound can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated NMU1 (or a polynucleotide encodingbiotinylated NMU1) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized inthe wells of streptavidin-coated plates (Pierce Chemical).Alternatively, antibodies which specifically bind to NMU1,polynucleotide, or a test compound, but which do not interfere with adesired binding site, such as the active site of NMU1, can bederivatized to the wells of the plate. Unbound target or protein can betrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodies whichspecifically bind to NMU1 polypeptide or test compound, enzyme-linkedassays which rely on detecting an activity of NMU1 polypeptide, and SDSgel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a NMU1 polypeptide orpolynucleotide also can be carried out in an intact cell. Any cell whichcomprises a NMU1 polypeptide or polynucleotide can be used in acell-based assay system. A NMU1 polynucleotide can be naturallyoccurring in the cell or can be introduced using techniques such asthose described above. Binding of the test compound to NMU1 or apolynucleotide encoding NMU1 is determined as described above.

Functional Assays

Test compounds can be tested for the ability to increase or decreaseNMU1 activity of a NMU1 polypeptide. The NMU1 activity can be measured,for example, using methods described in the specific examples, below.NMU1 activity can be measured after contacting either a purified NMU1, acell membrane preparation, or an intact cell with a test compound. Atest compound which decreases NMU1 activity by at least about 10,preferably about 50, more preferably about 75, 90, or 100% is identifiedas a potential agent for decreasing NMU1 activity. A test compound whichincreases NMU1 activity by at least about 10, preferably about 50, morepreferably about 75, 90, or 100% is identified as a potential agent forincreasing NMU1 activity.

One such screening procedure involves the use of melanophores which aretransfected to express NMU1. Such a screening technique is described inPCT WO 92/01810 published Feb. 6, 1992. Thus, for example, such an assaymay be employed for screening for a compound which inhibits activationof the receptor polypeptide of the present invention by contacting themelanophore cells which encode the receptor with both the receptorligand and a compound to be screened. Inhibition of the signal generatedby the ligand indicates that a compound is a potential antagonist forthe receptor, i.e., inhibits activation of the receptor. The screen maybe employed for identifying a compound which activates the receptor bycontacting such cells with compounds to be screened and determiningwhether each compound generates a signal, i.e., activates the receptor.

Other screening techniques include the use of cells which express NMU1(for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation [Iwabuchi,(1993)]. For example, compounds may be contacted with a cell whichexpresses the receptor polypeptide of the present invention and a secondmessenger response, e.g., signal transduction or pH changes, can bemeasured to determine whether the potential compound activates orinhibits the receptor. Another such screening technique involvesintroducing RNA encoding NMU1 into Xenopus oocytes to transientlyexpress the receptor. The receptor oocytes can then be contacted withthe receptor ligand and a compound to be screened, followed by detectionof inhibition or activation of a calcium signal in the case of screeningfor compounds which are thought to inhibit activation of the receptor.

Another screening technique involves expressing NMU1 in cells in whichthe receptor is linked to a phospholipase C or D. Such cells includeendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening may be accomplished as described above by quantifying thedegree of activation of the receptor from changes in the phospholipaseactivity.

Gene Expression

In another embodiment, test compounds which increase or decrease NMU1gene expression are identified. As used herein, the term “correlateswith expression of a polynucleotide” indicates that the detection of thepresence of nucleic acids, the same or related to a nucleic acidsequence encoding NMU1, by northern analysis or relatime PCR isindicative of the presence of nucleic acids encoding NMU1 in a sample,and thereby correlates with expression of the transcript from thepolynucleotide encoding NMU1. The term “microarray,” as used herein,refers to an array of distinct polynucleotides or oligonucleotidesarrayed on a substrate, such as paper, nylon or any other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport. A NMU1 polynucleotide is contacted with a test compound, andthe expression of an RNA or polypeptide product of NMU1 polynucleotideis determined. The level of expression of appropriate mRNA orpolypeptide in the presence of the test compound is compared to thelevel of expression of mRNA or polypeptide in the absence of the testcompound. The test compound can then be identified as a regulator ofexpression based on this comparison. For example, when expression ofmRNA or polypeptide is greater in the presence of the test compound thanin its absence, the test compound is identified as a stimulator orenhancer of the mRNA or polypeptide expression. Alternatively, whenexpression of the mRNA or polypeptide is less in the presence of thetest compound than in its absence, the test compound is identified as aninhibitor of the mRNA or polypeptide expression.

The level of NMU1 mRNA or polypeptide expression in the cells can bedetermined by methods well known in the art for detecting mRNA orpolypeptide. Either qualitative or quantitative methods can be used. Thepresence of polypeptide products of NMU1 polynucleotide can bedetermined, for example, using a variety of techniques known in the art,including immunochemical methods such as radioimmunoassay, Westernblotting, and immunohistochemistry. Alternatively, polypeptide synthesiscan be determined in vivo, in a cell culture, or in an in vitrotranslation system by detecting incorporation of labeled amino acidsinto NMU1.

Such screening can be carried out either in a cell-free assay system orin an intact cell. Any cell which expresses NMU1 polynucleotide can beused in a cell-based assay system. The NMU1 polynucleotide can benaturally occurring in the cell or can be introduced using techniquessuch as those described above. Either a primary culture or anestablished cell line can be used.

Test Compounds

Suitable test compounds for use in the screening assays of the inventioncan be obtained from any suitable source, e.g., conventional compoundlibraries. The test compounds can also be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds [Lam, (1997)]. Examples of methods forthe synthesis of molecular libraries can be found in the art. Librariesof compounds may be presented in solution or on beads, bacteria, spores,plasmids or phage.

Modeling of Regulators

Computer modeling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate NMU1 expression or activity. Having identified such a compoundor composition, the active sites or regions are identified. Such activesites might typically be ligand binding sites, such as the interactiondomain of the ligand with NMU1. The active site can be identified usingmethods known in the art including, for example, from the amino acidsequences of peptides, from the nucleotide sequences of nucleic acids,or from study of complexes of the relevant compound or composition withits natural ligand. In the latter case, chemical or X-raycrystallographic methods can be used to find the active site by findingwhere on the factor the complexed ligand is found.

Next, the three dimensional geometric structure of the active site isdetermined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intramolecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modeling can be used to complete thestructure or improve its accuracy. Any recognized modeling method may beused, including parameterized models specific to particular biopolymerssuch as proteins or nucleic acids, molecular dynamics models based oncomputing molecular motions, statistical mechanics models based onthermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a search can be manual, but is preferably computer assisted. Thesecompounds found from this search are potential NMU1 modulatingcompounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modeling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Therapeutic Indications and Methods

It was found by the present applicant that NMU1 is expressed in varioushuman tissues.

Central Nervous System (CNS) Disorders

CNS disorders include disorders of the central nervous system as well asdisorders of the peripheral nervous system.

CNS disorders include, but are not limited to brain injuries,cerebrovascular diseases and their consequences, Parkinson's disease,corticobasal degeneration, motor neuron disease, dementia, includingALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke,post-traumatic brain injury, and small-vessel cerebrovascular disease.Dementias, such as Alzheimer's disease, vascular dementia, dementia withLewy bodies, frontotemporal dementia and Parkinsonism linked tochromosome 17, frontotemporal dementias, including Pick's disease,progressive nuclear palsy, corticobasal degeneration, Huntington'sdisease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia,schizophrenia with dementia, and Korsakoff's psychosis, within themeaning of the invention are also considered to be CNS disorders.

Similarly, cognitive-related disorders, such as mild cognitiveimpairment, age-associated memory impairment, age-related cognitivedecline, vascular cognitive impairment, attention deficit disorders,attention deficit hyperactivity disorders, and memory disturbances inchildren with learning disabilities are also considered to be CNSdisorders.

Pain, within the meaning of the invention, is also considered to be aCNS disorder. Pain can be associated with CNS disorders, such asmultiple sclerosis, spinal cord injury, sciatica, failed back surgerysyndrome, traumatic brain injury, epilepsy, Parkinson's disease,post-stroke, and vascular lesions in the brain and spinal cord (e.g.,infarct, hemorrhage, vascular malformation). Non-central neuropathicpain includes that associated with post mastectomy pain, phantomfeeling, reflex sympathetic dystrophy (RSD), trigeminalneuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain,cancer pain, metabolic neuropathies (e.g., diabetic neuropathy,vasculitic neuropathy secondary to connective tissue disease),paraneoplastic polyneuropathy associated, for example, with carcinoma oflung, or leukemia, or lymphoma, or carcinoma of prostate, colon orstomach, trigeminal neuralgia, cranial neuralgias, and post-herpeticneuralgia. Pain associated with peripheral nerve damage, central pain(i.e. due to cerebral ischemia) and various chronic pain i.e., lumbago,back pain (low back pain), inflammatory and/or rheumatic pain. Headachepain (for example, migraine with aura, migraine without aura, and othermigraine disorders), episodic and chronic tension-type headache,tension-type like headache, cluster headache, and chronic paroxysmalhemicrania are also CNS disorders. Visceral pain such as pancreatits,intestinal cystitis, dysmenorrhea, irritable Bowel syndrome, Crohn'sdisease, biliary colic, ureteral colic, myocardial infarction and painsyndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethralsyndrome and protatodynia are also CNS disorders. Also considered to bea disorder of the nervous system are acute pain, for examplepostoperative pain, and pain after trauma.

NMU1 is highly expressed in various brain tissues. The expression inbrain tissues suggests an association of NMU1 with nervous systemdiseases. The expression of NMU1 receptor in brain in the state ofalzheimer is 10 fold higher than in healthy brain. NMU1 can be used totreat or diagnose diseases of the nervous system.

Cardiovascular Disorders

Heart failure is defined as a pathophysiological state in which anabnormality of cardiac function is responsible for the failure of theheart to pump blood at a rate commensurate with the requirement of themetabolizing tissue. It includes all forms of pumping failures such ashigh-output and low-output, acute and chronic, right-sided orleft-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (MI) is generally caused by an abrupt decrease incoronary blood flow that follows a thrombotic occlusion of a coronaryartery previously narrowed by arteriosclerosis. MI prophylaxis (primaryand secondary prevention) is included as well as the acute treatment ofMI and the prevention of complications.

Ischemic diseases are conditions in which the coronary flow isrestricted resulting in a perfusion which is inadequate to meet themyocardial requirement for oxygen. This group of diseases includesstable angina, unstable angina and asymptomatic ischemia.

Arrhythmias include all forms of atrial and ventriculartachyarrhythmias, atrial tachycardia, atrial flutter, atrialfibrillation, atrio-ventricular reentrant tachycardia, preexitationsyndrome, ventricular tachycardia, ventricular flutter, ventricularfibrillation, as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds ofsecondary arterial hypertension, renal, endocrine, neurogenic, others.The genes may be used as drug targets for the treatment of hypertensionas well as for the prevention of all complications arising fromcardiovascular diseases.

Peripheral vascular diseases are defined as vascular diseases in whicharterial and/or venous flow is reduced resulting in an imbalance betweenblood supply and tissue oxygen demand. It includes chronic peripheralarterial occlusive disease (PAOD), acute arterial thrombosis andembolism, inflammatory vascular disorders, Raynaud's phenomenon andvenous disorders.

Atherosclerosis is a cardiovascular disease in which the vessel wall isremodeled, compromising the lumen of the vessel. The atheroscleroticremodeling process involves accumulation of cells, both smooth musclecells and monocyte/macrophage inflammatory cells, in the intima of thevessel wall. These cells take up lipid, likely from the circulation, toform a mature atherosclerotic lesion. Although the formation of theselesions is a chronic process, occurring over decades of an adult humanlife, the majority of the morbidity associated with atherosclerosisoccurs when a lesion ruptures, releasing thrombogenic debris thatrapidly occludes the artery. When such an acute event occurs in thecoronary artery, myocardial infarction can ensue, and in the worst case,can result in death.

The formation of the atherosclerotic lesion can be considered to occurin five overlapping stages such as migration, lipid accumulation,recruitment of inflammatory cells, proliferation of vascular smoothmuscle cells, and extracellular matrix deposition. Each of theseprocesses can be shown to occur in man and in animal models ofatherosclerosis, but the relative contribution of each to the pathologyand clinical significance of the lesion is unclear.

Thus, a need exists for therapeutic methods and agents to treatcardiovascular pathologies, such as atherosclerosis and other conditionsrelated to coronary artery disease.

Cardiovascular diseases include but are not limited to disorders of theheart and the vascular system like congestive heart failure, myocardialinfarction, ischemic diseases of the heart, all kinds of atrial andventricular arrhythmias, hypertensive vascular diseases, peripheralvascular diseases, and atherosclerosis.

NMU1 is highly expressed in different cardiovascular related tissuessuch as pericardium and coronary artery smooth muscle cells. Expressionin the above mentioned tissues suggests an association between NMU1 andcardiovascular diseases. NMU1 can be regulated and measured in order totreat or diagnose cardiovascular diseases.

Hematological Disorders

Hematological disorders comprise diseases of the blood and all itsconstituents as well as diseases of organs involved in the generation ordegradation of the blood. They include but are not limited to 1)Anemias, 2) Myeloproliferative Disorders, 3) Hemorrhagic Disorders, 4)Leukopenia, 5) Eosinophilic Disorders, 6) Leukemias, 7) Lymphomas, 8)Plasma Cell Dyscrasias, 9) Disorders of the Spleen in the course ofhematological disorders, Disorders according to 1) include, but are notlimited to anemias due to defective or deficient hem synthesis,deficient erythropoiesis. Disorders according to 2) include, but are notlimited to polycythemia vera, tumor-associated erythrocytosis,myelofibrosis, thrombocythemia. Disorders according to 3) include, butare not limited to vasculitis, thrombocytopenia, heparin-inducedthrombocytopenia, thrombotic thrombocytopenic purpura, hemolytic-uremicsyndrome, hereditary and aquired disorders of platelet function,hereditary coagulation disorders. Disorders according to 4) include, butare not limited to neutropenia, lymphocytopenia. Disorders according to5) include, but are not limited to hypereosinophilia, idiopathichypereosinophilic syndrome. Disorders according to 6) include, but arenot limited to acute myeloic leukemia, acute lymphoblastic leukemia,chronic myelocytic leukemia, chronic lymphocytic leukemia,myelodysplastic syndrome. Disorders according to 7) include, but are notlimited to Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt'slymphoma, mycosis fungoides cutaneous T-cell lymphoma. Disordersaccording to 8) include, but are not limited to multiple myeloma,macroglobulinemia, heavy chain diseases. In extension of the precedingidiopathic thrombocytopenic purpura, iron deficiency anemia,megaloblastic anemia (vitamin B12 deficiency), aplastic anemia,thalassemia, malignant lymphoma bone marrow invasion, malignant lymphomaskin invasion, haemolytic uraemic syndrome, giant platelet disease areconsidered to be hematological diseases too.

NMU1 is highly expressed in leukocytes and other tissues of thehematological system. The expression in the above mentioned tissuessuggests an association between NMU1 and hematological diseases. NMU1can be regulated and measured in order to treat or to diagnosehematological disorders.

Inflammatory Diseases

Inflammatory diseases comprise diseases triggered by cellular ornon-cellular mediators of the immune system or tissues causing theinflammation of body tissues and subsequently producing an acute orchronic inflammatory condition. Examples for such inflammatory diseasesare hypersensitivity reactions of type I-IV, for example but not limitedto hypersensitivity diseases of the lung including asthma, atopicdiseases, allergic rhinitis or conjunctivitis, angioedema of the lids,hereditary angioedema, antireceptor hypersensitivity reactions andautoimmune diseases, Hashimoto's thyroiditis, systemic lupuserythematosus, Goodpasture's syndrome, pemphigus, myasthenia gravis,Grave's and Raynaud's disease, type B insulin-resistant diabetes,rheumatoid arthritis, psoriasis, Crohn's disease, scleroderma, mixedconnective tissue disease, polymyositis, sarcoidosis,glomerulonephritis, acute or chronic host versus graft reactions.

The NMU 1 receptor is highly expressed in different tissues of theimmune system and tissues responsive to components of the immune systemas well as tissues responsive to mediators of inflammation. Theexpression in the above mentioned tissues suggests an associationbetween NMU1 and inflammatory diseases. NMU1 can be regulated to treatinflammatory diseases and NMU 1 can be measured in order to diagnosesuch diseases.

Liver Diseases

Liver diseases comprise primary or secondary, acute or chronic diseasesor injury of the liver which may be acquired or inherited, benign ormalignant, and which may affect the liver or the body as a whole. Theycomprise but are not limited to disorders of the bilirubin metabolism,jaundice, syndroms of Gilbert's, Crigler-Najjar, Dubin-Johnson andRotor; intrahepatic cholestasis, hepatomegaly, portal hypertension,ascites, Budd-Chiari syndrome, portal-systemic encephalopathy, fattyliver, steatosis, Reye's syndrome, liver diseases due to alcohol,alcoholic hepatitis or cirrhosis, fibrosis and cirrhosis, fibrosis andcirrhosis of the liver due to inborn errors of metabolism or exogenoussubstances, storage diseases, syndromes of Gaucher's, Zellweger's,Wilson's—disease, acute or chronic hepatitis, viral hepatitis and itsvariants, inflammatory conditions of the liver due to viruses, bacteria,fingi, protozoa, helminths; drug induced disorders of the liver, chronicliver diseases like primary sclerosing cholangitis,alpha₁-antitrypsin-deficiency, primary biliary cirrhosis, postoperativeliver disorders like postoperative intrahepatic cholestasis, hepaticgranulomas, vascular liver disorders associated with systemic disease,benign or malignant neoplasms of the liver, disturbance of livermetabolism in the new-born or prematurely born.

The NMU 1 receptor is highly expressed in liver tissues. The expressionin liver tissues suggests an association between NMU1 and liverdiseases. NMU1 can be regulated to treat liver diseases and NMU 1 can bemeasured to diagnose such diseases.

Cancer Disorders

Cancer disorders within the scope of the invention comprise any diseaseof an organ or tissue in mammals characterized by poorly controlled oruncontrolled multiplication of normal or abnormal cells in that tissueand its effect on the body as a whole. Cancer diseases within the scopeof the invention comprise benign neoplasms, dysplasias, hyperplasias aswell as neoplasms showing metastatic growth or any other transformationslike e.g. leukoplakias which often precede a breakout of cancer. Cellsand tissues are cancerous when they grow more rapidly than normal cells,displacing or spreading into the surrounding healthy tissue or any othertissues of the body described as metastatic growth, assume abnormalshapes and sizes, show changes in their nucleocytoplasmatic ratio,nuclear polychromasia, and finally may cease. Cancerous cells andtissues may affect the body as a whole when causing paraneoplasticsyndromes or if cancer occurs within a vital organ or tissue, normalfunction will be impaired or halted, with possible fatal results. Theultimate involvement of a vital organ by cancer, either primary ormetastatic, may lead to the death of the mammal affected. Cancer tendsto spread, and the extent of its spread is usually related to anindividual's chances of surviving the disease. Cancers are generallysaid to be in one of three stages of growth: early, or localized, when atumor is still confined to the tissue of origin, or primary site; directextension, where cancer cells from the tumour have invaded adjacenttissue or have spread only to regional lymph nodes; or metastasis, inwhich cancer cells have migrated to distant parts of the body from theprimary site, via the blood or lymph systems, and have establishedsecondary sites of infection. Cancer is said to be malignant because ofits tendency to cause death if not treated. Benign tumors usually do notcause death, although they may if they interfere with a normal bodyfunction by virtue of their location, size, or paraneoplastic sideeffects. Hence benign tumors fall under the definition of cancer withinthe scope of the invention as well. In general, cancer cells divide at ahigher rate than do normal cells, but the distinction between the growthof cancerous and normal tissues is not so much the rapidity of celldivision in the former as it is the partial or complete loss of growthrestraint in cancer cells and their failure to differentiate into auseful, limited tissue of the type that characterizes the functionalequilibrium of growth of normal tissue. Cancer tissues may expresscertain molecular receptors and probably are influenced by the host'ssusceptibility and immunity and it is known that certain cancers of thebreast and prostate, for example, are considered dependent on specifichormones for their existence. The term “cancer” under the scope of theinvention is not limited to simple benign neoplasia but comprises anyother benign and malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3)Carcinosarcoma, 4) Cancers of the blood-forming tissues, 5) tumors ofnerve tissues including the brain, 6) cancer of skin cells. Canceraccording to 1) occurs in epithelial tissues, which cover the outer body(the skin) and line mucous membranes and the inner cavitary structuresof organs e.g. such as the breast, lung, the respiratory andgastrointestinal tracts, the endocrine glands, and the genitourinarysystem. Ductal or glandular elements may persist in epithelial tumors,as in adenocarcinomas like e.g. thyroid adenocarcinoma, gastricadenocarcinoma, uterine adenocarcinoma. Cancers of the pavement-cellepithelium of the skin and of certain mucous membranes, such as e.g.cancers of the tongue, lip, larynx, urinary bladder, uterine cervix, orpenis, may be termed epidermoid or squamous-cell carcinomas of therespective tissues and are in the scope of the definition of cancer aswell. Cancer according to 2) develops in connective tissues, includingfibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, andcartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma,synovial sarcoma. Cancer according to 3) is cancer that develops in bothepithelial and connective tissue. Cancer disease within the scope ofthis definition may be primary or secondary, whereby primary indicatesthat the cancer originated in the tissue where it is found rather thanwas established as a secondary site through metastasis from anotherlesion. Cancers and tumor diseases within the scope of this definitionmay be benign or malign and may affect all anatomical structures of thebody of a mammal. By example but not limited to they comprise cancersand tumor diseases of I) the bone marrow and bone marrow derived cells(leukemias), II) the endocrine and exocrine glands like e.g. thyroid,parathyroid, pituitary, adrenal glands, salivary glands, pancreas I) thebreast, like e.g. benign or malignant tumors in the mammary glands ofeither a male or a female, the mammary ducts, adenocarcinoma, medullarycarcinoma, comedo carcinoma, Paget's disease of the nipple, inflammatorycarcinoma of the young woman, IV) the lung, V) the stomach, VI) theliver and spleen, VII) the small intestine, VIII) the colon, IX) thebone and its supportive and connective tissues like malignant or benignbone tumour, e.g. malignant osteogenic sarcoma, benign osteoma,cartilage tumors; like malignant chondrosarcoma or benign chondroma;bone marrow tumors like malignant myeloma or benign eosinophilicgranuloma, as well as metastatic tumors from bone tissues at otherlocations of the body; X) the mouth, throat, larynx, and the esophagus,XI) the urinary bladder and the internal and external organs andstructures of the urogenital system of male and female like ovaries,uterus, cervix of the uterus, testes, and prostate gland, XII) theprostate, XIII) the pancreas, like ductal carcinoma of the pancreas;XIV) the lymphatic tissue like lymphomas and other tumors of lymphoidorigin, XV) the skin, XVI) cancers and tumor diseases of all anatomicalstructures belonging to the respiration and respiratory systemsincluding thoracal muscles and linings, XVII) primary or secondarycancer of the lymph nodes XVIII) the tongue and of the bony structuresof the hard palate or sinuses, XVIV) the mouth, cheeks, neck andsalivary glands, XX) the blood vessels including the heart and theirlinings, XXI) the smooth or skeletal muscles and their ligaments andlinings, XXII) the peripheral, the autonomous, the central nervoussystem including the cerebellum, XXIII) the adipose tissue.

The NMU 1 receptor is highly expressed in different cancer tissues suchas colon cancer and lung cancer. The expression in the above mentionedtissues suggests an association between NMU1 and cancer. NMU1 can beregulated and measured in order to diagnose and treat cancer.

Applications

The present invention provides for both prophylactic and therapeuticmethods for hematological diseases, cardiovascular diseases, disordersof the peripheral and central nervous system, inflammation diseases,cancer diseases, and disorders of the liver.

The regulatory method of the invention involves contacting a cell withan agent that modulates one or more of the activities of NMU1. An agentthat modulates activity can be an agent as described herein, such as anucleic acid or a protein, a naturally-occurring cognate ligand of thepolypeptide, a peptide, a peptidominmetic, or any small molecule. In oneembodiment, the agent stimulates one or more of the biologicalactivities of NMU1. Examples of such stimulatory agents include theactive NMU1 and nucleic acid molecules encoding a portion of NMU1. Inanother embodiment, the agent inhibits one or more of the biologicalactivities of NMU1. Examples of such inhibitory agents include antisensenucleic acid molecules and antibodies. These regulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g, by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byunwanted expression or activity of NMU1 or a protein in the NMU1signaling pathway. In one embodiment, the method involves administeringan agent like any agent identified or being identifiable by a screeningassay as described herein, or combination of such agents that modulatesay upregulate or downregulate the expression or activity of NMU1 or ofany protein in the NMU1 signaling pathway. In another embodiment, themethod involves administering a regulator of NMU1 as therapy tocompensate for reduced or undesirably low expression or activity of NMU1or a protein in the NMU1 signaling pathway.

Stimulation of activity or expression of NMU1 is desirable in situationsin which activity or expression is abnormally low and in which increasedactivity is likely to have a beneficial effect. Conversely, inhibitionof activity or expression of NMU1 is desirable in situations in whichactivity or expression of NMU1 is abnormally high and in whichdecreasing its activity is likely to have a beneficial effect.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

Pharmaceutical Compositions

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

The nucleic acid molecules, polypeptides, and antibodies (also referredto herein as “active compounds”) of the invention can be incorporatedinto pharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The invention includes pharmaceutical compositions comprising aregulator of NMU1 expression or activity (and/or a regulator of theactivity or expression of a protein in the NMU1 signaling pathway) aswell as methods for preparing such compositions by combining one or moresuch regulators and a pharmaceutically acceptable carrier. Also withinthe invention are pharmaceutical compositions comprising a regulatoridentified using the screening assays of the invention packaged withinstructions for use. For regulators that are antagonists of NMU1activity or which reduce NMU1 expression, the instructions would specifyuse of the pharmaceutical composition for treatment of hematological andcardiovascular diseases, disorders of the peripheral and central nervoussystem, COPD, asthma, genito-urological disorders and inflammationdiseases. For regulators that are agonists of NMU1 activity or increaseNMU1 expression, the instructions would specify use of thepharmaceutical composition for treatment of hematological andcardiovascular diseases, disorders of the peripheral and central nervoussystem, COPD, asthma, genito-urological disorders and inflammationdiseases.

An antagonist of NMU1 may be produced using methods which are generallyknown in the art. In particular, purified NMU1 may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind NMU1. Antibodies to NMU1 may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,single chain antibodies, Fab fragments, and fragments produced by a Fabexpression library. Neutralizing antibodies like those which inhibitdimer formation are especially preferred for therapeutic use.

In another embodiment of the invention, the polynucleotides encodingNMU1, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingNMU1 may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding NMU1. Thus,complementary molecules or fragments may be used to modulate NMU1activity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding NMU1.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencecomplementary to the polynucleotides of the gene encoding NMU1. Thesetechniques are described, for example, in [Scott and Smith (1990)Science 249:386-390].

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition containing NMU1 in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of NMU1,antibodies to NMU1, and mimetics, agonists, antagonists, or inhibitorsof NMU1. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fingi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyetheylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin. Sterileinjectable solutions can be prepared by incorporating the activecompound (e.g., a polypeptide or antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. Forpharmaceutical compositions which include an antagonist of NMU1activity, a compound which reduces expression of NMU1, or a compoundwhich reduces expression or activity of a protein in the NMU1 signalingpathway or any combination thereof, the instructions for administrationwill specify use of the composition for hematological and cardiovasculardiseases, disorders of the peripheral and central nervous system, COPD,asthma, genito-urological disorders and inflammation diseases. Forpharmaceutical compositions which include an agonist of NMU1 activity, acompound which increases expression of NMU1, or a compound whichincreases expression or activity of a protein in the NMU1 signalingpathway or any combination thereof, the instructions for administrationwill specify use of the composition for hematological and cardiovasculardiseases, disorders of the peripheral and central nervous system, COPD,asthma, genito-urological disorders and inflammation diseases.

Diagnostics

In another embodiment, antibodies which specifically bind NMU1 may beused for the diagnosis of disorders characterized by the expression ofNMU1, or in assays to monitor patients being treated with NMU1 oragonists, antagonists, and inhibitors of NMU1. Antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for NMU1 includemethods which utilize the antibody and a label to detect NMU1 in humanbody fluids or in extracts of cells or tissues. The antibodies may beused with or without modification, and may be labeled by covalent ornon-covalent joining with a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring NMU1, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of NMU1 expression. Normal or standard values for NMU1expression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toNMU1 under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods,preferably by photometric means. Quantities of NMU1 expressed in subjectsamples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingNMU1 may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofNMU1 may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of NMU1,and to monitor regulation of NMU1 levels during therapeuticintervention.

Polynucleotide sequences encoding NMU1 may be used for the diagnosis ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver associated with expression of NMU1.The polynucleotide sequences encoding NMU1 may be used in Southern,Northern, or dot-blot analysis, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patient biopsies to detect altered NMU1expression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding NMU1 may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingNMU1 may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered from that of a comparable control sample, the nucleotidesequences have hybridized with nucleotide sequences in the sample, andthe presence of altered levels of nucleotide sequences encoding NMU1 inthe sample indicates the presence of the associated disorder. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orin monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver associated with expression of NMU1, a normal or standard profilefor expression is established. This may be accomplished by combiningbody fluids or cell extracts taken from normal subjects, either animalor human, with a sequence, or a fragment thereof, encoding NMU1, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained from normal samples may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with NMU1, orfragments thereof, and washed. Bound NMU1 is then detected by methodswell known in the art. Purified NMU1 can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding NMU1 specificallycompete with a test compound for binding NMU1. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with NMU1.

G-protein coupled receptors are ubiquitous in the mammalian host and areresponsible for many biological functions, including many pathologies.Accordingly, it is desirable to find compounds and drugs which stimulatea G-protein coupled receptor on the one hand and which can inhibit thefunction of a G-protein coupled receptor on the other hand. For example,compounds which activate the G-protein coupled receptor may be employedfor therapeutic purposes, such as the treatment of asthma, Parkinson'sdisease, acute heart failure, urinary retention, and osteoporosis. Inparticular, compounds which activate the receptors of the presentinvention are useful in treating various cardiovascular ailments such ascaused by the lack of pulmonary blood flow or hypertension. In additionthese compounds may also be used in treating various physiologicaldisorders relating to abnormal control of fluid and electrolytehomeostasis and in diseases associated with abnormal angiotensin-inducedaldosterone secretion.

In general, compounds which inhibit activation of the G-protein coupledreceptor may be employed for a variety of therapeutic purposes, forexample, for the treatment of hypotension and/or hypertension, anginapectoris, myocardial infarction, ulcers, asthma, allergies, benignprostatic hypertrophy, and psychotic and neurological disordersincluding schizophrenia, manic excitement, depression, delirium,dementia or severe mental retardation, dyskinesias, such as Huntington'sdisease or Tourett's syndrome, among others. Compounds which inhibitG-protein coupled receptors have also been useful in reversingendogenous anorexia and in the control of bulimia.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to that amount of active ingredient which increases or decreasesNMU1 activity relative to NMU1 activity which occurs in the absence ofthe therapeutically effective dose. For any compound, thetherapeutically effective dose can be estimated initially either in cellculture assays or in animal models, usually mice, rabbits, dogs, orpigs. The animal model also can be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population), can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration. The exact dosage will be determined by thepractitioner, in light of factors related to the subject that requirestreatment. Dosage and administration are adjusted to provide sufficientlevels of the active ingredient or to maintain the desired effect.Factors which can be taken into account include the severity of thedisease state, general health of the subject, age, weight, and gender ofthe subject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long-acting pharmaceutical compositions can be administeredevery 3 to 4 days, every week, or once every two weeks depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 micrograms to 100,000micrograms, up to a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc. If the reagent is asingle-chain antibody, polynucleotides encoding the antibody can beconstructed and introduced into a cell either ex vivo or in vivo usingwell-established techniques including, but not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and DEAE- orcalcium phosphate-mediated transfection.

If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides which expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above. Preferably, a reagent reducesexpression of NMU1 gene or the activity of NMU1 by at least about 10,preferably about 50, more preferably about 75, 90, or 100% relative tothe absence of the reagent. The effectiveness of the mechanism chosen todecrease the level of expression of NMU1 gene or the activity of NMU1can be assessed using methods well known in the art, such ashybridization of nucleotide probes to NMU1-specific mRNA, quantitativeRT-PCR, immunologic detection of NMU1, or measurement of NMU1 activity.

In any of the embodiments described above, any of the pharmaceuticalcompositions of the invention can be administered in combination withother appropriate therapeutic agents. Selection of the appropriateagents for use in combination therapy can be made by one of ordinaryskill in the art, according to conventional pharmaceutical principles.The combination of therapeutic agents can act synergistically to effectthe treatment or prevention of the various disorders described above.Using this approach, one may be able to achieve therapeutic efficacywith lower dosages of each agent, thus reducing the potential foradverse side effects. Any of the therapeutic methods described above canbe applied to any subject in need of such therapy, including, forexample, mammals such as dogs, cats, cows, horses, rabbits, monkeys, andmost preferably, humans.

Nucleic acid molecules of the invention are those nucleic acid moleculeswhich are contained in a group of nucleic acid molecules consisting of(i) nucleic acid molecules encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, (ii) nucleic acid molecules comprisingthe sequence of SEQ ID NO: 1, (iii) nucleic acid molecules having thesequence of SEQ ID NO: 1, (iv) nucleic acid molecules the complementarystrand of which hybridizes under stringent conditions to a nucleic acidmolecule of (i), (ii), or (iii); and (v) nucleic acid molecules thesequence of which differs from the sequence of a nucleic acid moleculeof (iii) due to the degeneracy of the genetic code, wherein thepolypeptide encoded by said nucleic acid molecule has NMU1 activity.

Polypeptides of the invention are those polypeptides which are containedin a group of polypeptides consisting of (i) polypeptides having thesequence of SEQ ID NO: 2, (ii) polypeptides comprising the sequence ofSEQ ID NO: 2, (iii) polypeptides encoded by nucleic acid molecules ofthe invention and (iv) polypeptides which show at least 99%, 98%, 95%,90%, or 80% homology with a polypeptide of (i), (ii), or (iii), whereinsaid purified polypeptide has NMU1 activity.

An object of the invention is a method of screening for therapeuticagents useful in the treatment of a disease comprised in a group ofdiseases consisting of hematological diseases, cardiovascular diseases,disorders of the peripheral and central nervous system, inflammationdiseases, cancer diseases, and disorders of the liver in a mammalcomprising the steps of (i) contacting a test compound with a NMU1polypeptide, (ii) detect binding of said test compound to said NMU1polypeptide. E.g., compounds that bind to the NMU1 polypeptide areidentified potential therapeutic agents for such a disease.

Another object of the invention is a method of screening for therapeuticagents useful in the treatment of a disease comprised in a group ofdiseases consisting of hematological diseases, cardiovascular diseases,disorders of the peripheral and central nervous system, inflammationdiseases, cancer diseases, and disorders of the liver in a mammalcomprising the steps of (i) determining the activity of a NMU1polypeptide at a certain concentration of a test compound or in theabsence of said test compound, (ii) determining the activity of saidpolypeptide at a different concentration of said test compound. E.g.,compounds that lead to a difference in the activity of the NMU1polypeptide in (i) and (ii) are identified potential therapeutic agentsfor such a disease.

Another object of the invention is a method of screening for therapeuticagents useful in the treatment of a disease comprised in a group ofdiseases consisting of hematological diseases, cardiovascular diseases,disorders of the peripheral and central nervous system, inflammationdiseases, cancer diseases, and disorders of the liver in a mammalcomprising the steps of (i) determining the activity of a NMU1polypeptide at a certain concentration of a test compound, (ii)determining the activity of a NMU1 polypeptide at the presence of acompound known to be a regulator of a NMU1 polypeptide. E.g., compoundsthat show similar effects on the activity of the NMU1 polypeptide in (i)as compared to compounds used in (ii) are identified potentialtherapeutic agents for such a disease.

Other objects of the invention are methods of the above, wherein thestep of contacting is in or at the surface of a cell.

Other objects of the invention are methods of the above, wherein thecell is in vitro.

Other objects of the invention are methods of the above, wherein thestep of contacting is in a cell-free system.

Other objects of the invention are methods of the above, wherein thepolypeptide is coupled to a detectable label.

Other objects of the invention are methods of the above, wherein thecompound is coupled to a detectable label.

Other objects of the invention are methods of the above, wherein thetest compound displaces a ligand which is first bound to thepolypeptide.

Other objects of the invention are methods of the above, wherein thepolypeptide is attached to a solid support.

Other objects of the invention are methods of the above, wherein thecompound is attached to a solid support.

Another object of the invention is a method of screening for therapeuticagents useful in the treatment of a disease comprised in a group ofdiseases consisting of hematological diseases, cardiovascular diseases,disorders of the peripheral and central nervous system, inflammationdiseases, cancer diseases, and disorders of the liver in a mammalcomprising the steps of (i) contacting a test compound with a NMU1polynucleotide, (ii) detect binding of said test compound to said NMU1polynucleotide. Compounds that, e.g., bind to the NMU1 polynucleotideare potential therapeutic agents for the treatment of such diseases.

Another object of the invention is the method of the above, wherein thenucleic acid molecule is RNA.

Another object of the invention is a method of the above, wherein thecontacting step is in or at the surface of a cell.

Another object of the invention is a method of the above, wherein thecontacting step is in a cell-free system.

Another object of the invention is a method of the above, wherein thepolynucleotide is coupled to a detectable label.

Another object of the invention is a method of the above, wherein thetest compound is coupled to a detectable label.

Another object of the invention is a method of diagnosing a diseasecomprised in a group of diseases consisting of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver in a mammal comprising the steps of (i) determining the amount ofa NMU1 polynucleotide in a sample taken from said mammal, (ii)determining the amount of NMU1 polynucleotide in healthy and/or diseasedmammal. A disease is diagnosed, e.g., if there is a substantialsimilarity in the amount of NMU1 polynucleotide in said test mammal ascompared to a diseased mammal.

Another object of the invention is a pharmaceutical composition for thetreatment of a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising atherapeutic agent which binds to a NMU1 polypeptide.

Another object of the invention is a pharmaceutical composition for thetreatment of a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising atherapeutic agent which regulates the activity of a NMU1 polypeptide.

Another object of the invention is a pharmaceutical composition for thetreatment of a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising atherapeutic agent which regulates the activity of a NMU1 polypeptide,wherein said therapeutic agent is (i) a small molecule, (ii) an RNAmolecule, (iii) an antisense oligonucleotide, (iv) a polypeptide, (v) anantibody, or (vi) a ribozyme.

Another object of the invention is a pharmaceutical composition for thetreatment of a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising a NMU1polynucleotide.

Another object of the invention is a pharmaceutical composition for thetreatment of a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising a NMU1polypeptide.

Another object of the invention is the use of regulators of a NMU1 forthe preparation of a pharmaceutical composition for the treatment of adisease comprised in a group of diseases consisting of hematologicaldiseases, cardiovascular diseases, disorders of the peripheral andcentral nervous system, inflammation diseases, cancer diseases, anddisorders of the liver in a mammal.

Another object of the invention is a method for the preparation of apharmaceutical composition useful for the treatment of a diseasecomprised in a group of diseases consisting of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver in a mammal comprising the steps of (i) identifying a regulator ofNMU1, (ii) determining whether said regulator ameliorates the symptomsof a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal; and (iii) combining ofsaid regulator with an acceptable pharmaceutical carrier.

Another object of the invention is the use of a regulator of NMU1 forthe regulation of NMU1 activity in a mammal having a disease comprisedin a group of diseases consisting of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention.

EXAMPLES Example 1 Search for Homologous Sequences in Public SequenceData Bases

The degree of homology can readily be calculated by known methods.Preferred methods to determine homology are designed to give the largestmatch between the sequences tested. Methods to determine homology arecodified in publicly available computer programs such as BestFit,BLASTP, BLASTN, and FASTA. The BLAST programs are publicly availablefrom NCBI and other sources in the internet.

For NMU1 the following hits to known sequences were identified by usingthe BLAST algorithm [Altschul S F, Madden T L, Schaffer A A, Zhang J,Zhang Z, Miller W, Lipman D J; Nucleic Acids Res 1997 Sep. 1; 25(17):3389-402] and the following set of parameters: matrix=BLOSUM62 and lowcomplexity filter. The following databases were searched: NCBI(non-redundant database) and DERWENT patent database (Geneseq).

The following hits were found:

-   >NA2001:AAD08007 Aad08007 Human G-protein coupled receptor, SNORF62,    cDNA. 8/2001, Length=1318, Score=2403 bits (1212), Expect=0.0,    Identities=1212/1212 (100%), frame: +1-   >NA2000:AAA30663 Aaa30663 Human G protein-coupled receptor MIG cDNA.    8/2000; Length=1212; Score=2403 bits (1212), Expect=0.0;    Identities=1212/1212 (100%); frame: +1-   >gb|AF272362.1|AF272362 Homo sapiens neuromedin U receptor 1 (NMUR1)    mRNA, complete cds; Length=1318; Score=2403 bits (1212), Expect=0.0;    Identities=1212/1212 (100%); frame: +1-   >ref|NM 006056.11 Homo sapiens G protein-coupled receptor 66    (GPR66), mRNA; Length=1212; Score=2403 bits (1212), Expect=0.0;    Identities=1212/1212 (100%); frame: +1-   >NA2001:AAF76231 Aaf76231 Human G-protein coupled receptor FM-3    cDNA. 6/2001; Length=1209; Score=2397 bits (1209), Expect=0.0;    Identities=1209/1209 (100%); frame: +1-   >NA2001:AAH45072 Aah45072 Human FM-3 coding sequence. 9/2001;    Length=1209; Score=2397 bits (1209), Expect=0.0;    Identities=1209/1209 (100%); frame: +1-   >NA2000:AAA30739 Aaa30739 DNA encoding human mutant G    protein-coupled receptor MIG (T273K). 8/2000; Length=1212;    Score=2387 bits (1204), Expect=0.0; Identities=1210/1212 (99%);    frame: +1-   >NA2000:AAZ49707 Aaz49707 Human growth hormone secretagogue related    receptor DNA. 4/2000; Length=1212; Score=2379 bits (1200),    Expect=0.0; Identities=1209/1212 (99%); frame: +1-   >gb|AC017104.8|Homo sapiens chromosome 2 clone RP11-56215, complete    sequence; Length=168880; Score=1643 bits (829), Expect=0.0;    Identities=829/829 (100%); frame+1-   >gb|AF044600.1|HSOGPCR1 Homo sapiens orphan G protein-coupled    receptor gene, first coding exon; Length=828; Score=1641 bits (828),    Expect=0.0; Identities=828/828 (100%); frame+1-   >gb|AF044601.1|HSOGPCR2 Homo sapiens orphan G protein-coupled    receptor gene, second coding exon and complete cds; Length=384;    Score=761 bits (384), Expect=0.0; Identities=384/384 (100%); frame:    +1-   >NA2001:AAH45073 Aah45073 Murine FM-3 coding sequence. 9/2001;    Length=1215; Score=317 bits (160), Expect=8e-84; Identities=499/612    (81%); frame: +1-   >NA2000:AAZ49706 Aaz49706 Mouse growth hormone secretagogue related    receptor DNA. 4/2000; Length=1526; Score=317 bits (160),    Expect=8e-84; Identities=499/612 (81%); frame: +1

Example 2 Expression Profiling

Total cellular RNA was isolated from cells by one of two standardmethods: 1) guanidine isothiocyanate/Cesium chloride density gradientcentrifugation [Kellogg, (1990)]; or with the Tri-Reagent protocolaccording to the manufacturer's specifications (Molecular ResearchCenter, Inc., Cincinatti, Ohio). Total RNA prepared by the Tri-reagentprotocol was treated with DNAse I to remove genomic DNA contamination.

For relative quantitation of the mRNA distribution of NMU1, total RNAfrom each cell or tissue source was first reverse transcribed. 85 μg oftotal RNA was reverse transcribed using 1 μmole random hexamer primers,0.5 mM each of DATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680μl. The first strand synthesis buffer and Omniscript reversetranscriptase (2 u/μl) were from (Qiagen, Hilden, Germany). The reactionwas incubated at 37° C. for 90 minutes and cooled on ice. The volume wasadjusted to 6800 μl with water, yielding a final concentration of 12.5ng/μl of starting RNA.

For relative quantitation of the distribution of NMU1 mRNA in cells andtissues the Perkin Elmer ABI Prism RTM. 7700 Sequence Detection systemor Biorad iCycler was used according to the manufacturer'sspecifications and protocols. PCR reactions were set up to quantitateNMU1 and the housekeeping genes HPRT (hypoxanthinephosphoribosyltransferase), GAPDH (glyceraldehyde-3-phosphatedehydrogenase), β-actin, and others. Forward and reverse primers andprobes for NMU1 were designed using the Perkin Elmer ABI Primer Express™software and were synthesized by TibMolBiol (Berlin, Germany). The NMU1forward primer sequence was: Primer1 (SEQ ID NO: 3). The NMU1 reverseprimer sequence was Primer2 (SEQ ID NO: 5). Probe1 (SEQ ID NO: 4),labelled with FAM (carboxyfluorescein succinimidyl ester) as thereporter dye and TAMRA (carboxytetramethylrhodamine) as the quencher, isused as a probe for NMU1. The following reagents were prepared in atotal of 25 μl: 1×TaqMan buffer A, 5.5 mM MgCl₂, 200 nM of DATP, dCTP,dGTP, and dUTP, 0.025 U/μl AmpliTaq Gold™, 0.01 U/μl AmpErase and Probe1(SEQ ID NO: 4), NMU1 forward and reverse primers each at 200 nM, 200 nMNMU1 FAM/TAMRA-labelled probe, and 5 μl of template cDNA. Thermalcycling parameters were 2 min at 50° C., followed by 10 min at 95° C.,followed by 40 cycles of melting at 95° C. for 15 sec andannealing/extending at 60° C. for 1 min.

Calculation of Corrected CT Values

The CT (threshold cycle) value is calculated as described in the“Quantitative determination of nucleic acids” section. The CF-value(factor for threshold cycle correction) is calculated as follows:

-   1. PCR reactions were set up to quantitate the housekeeping genes    (HKG) for each cDNA sample.-   2. CT_(HKG)-values (threshold cycle for housekeeping gene) were    calculated as described in the “Quantitative determination of    nucleic acids” section.-   3. CT_(HKG)-mean values (CT mean value of all HKG tested on one    cDNAs) of all HKG for each cDNA are calculated (n=number of HKG):-   CT_(HKG-n)-mean value ═(CT_(HKG)-value+CT_(HKG2)-value+ . . .    +CT_(HKG-n)-value)/n-   4. CT_(pannel) mean value (CT mean value of all HKG in all tested    cDNAs)=(CT_(HKG1)-mean value+CT_(HKG2)-mean value+ . . .    +CT_(HKG-y)-mean value)/y (y=number of cDNAs)-   5. CF_(cDNA-n) (correction factor for cDNA n)=CT_(pannel)-mean    value−CT_(HKG-n)-mean value-   6. CT_(cDNA-n) (CT value of the tested gene for the cDNA    n)+CF_(cDNA-n) (correction factor for cDNA n)=CT_(cor-cDNA-n)    (corrected CT value for a gene on cDNA n)    Calculation of Relative Expression-   Definition: highest CT_(cor-cDNA-n)≠40 is defined as CT_(cor-cDNA)    [high] Relative Expression=2^((CTcor-cDNA[high]<CTcor-cDNA-n))    Tissues

The expression of NMU1 was investigated in the following tissues: brain,total Alzheimer brain, fetal brain, cerebellum, spinal cord, heart,fetal heart, pericardium, peripheral blood, coronary smooth musclecells, HUVEC cells, thyroid, thyroid tumor, spleen, spleen livercirrhosis, thymus, bone marrow, lung, fetal lung, lung tumor, liver,fetal liver, liver liver cirrhosis, HEP G2 cells, pancreas, pancreasliver cirrhosis, stomach, small intestine, colon, colon tumor, kidney,HEK 293 cells, skeletal muscle, HeLa cells, breast tumor, mammary gland,MDA MB 231 cells, testis, trachea, adrenal gland, salivary gland,bladder, prostata, placenta, uterus, adipose

Expression Profile

The results of the mRNA-quantification (expression profiling) is shownin Table 1. TABLE 1 Relative expression of NMU1 in various humantissues. tissue relative expression brain 24,45 total Alzheimer brain276,44  fetal brain 18,45 cerebellum 38,77 spinal cord  8,26 heart  2,18fetal heart 64,26 pericardium 160,25  peripheral blood 1318,79  coronarysmooth muscle cells 802,95  HUVEC cells 973,81  thyroid 307,44  thyroidtumor 42,44 spleen 61,62 spleen liver cirrhosis 146,27  thymus 53,48bone marrow 89,88 lung 54,13 fetal lung 97,06 lung tumor 469,23  liver759,64  fetal liver 17,97 liver cirrhosis 897,13  HEP G2 cells 622,03 pancreas 72,15 pancreas liver cirrhosis  9,90 stomach 161,13  smallintestine 290,89  colon 314,08  colon tumor 195,70  kidney 402,87  HEK293 cells 230,32  skeletal muscle 10,63 HeLa cells  1,00 breast tumor198,43  mammary gland 136,87  MDA MB 231 cells 31,83 testis 115,11 trachea 186,88  adrenal gland 57,67 salivary gland 10,21 bladder 162,77 prostata 207,52  placenta 156,74  uterus 285,97  adipose 156,77 

Example 3 Antisense Analysis

Knowledge of the correct, complete cDNA sequence coding for NMU1 enablesits use as a tool for antisense technology in the investigation of genefunction. Oligonucleotides, cDNA or genomic fragments comprising theantisense strand of a polynucleotide coding for NMU1 are used either invitro or in vivo to inhibit translation of the mRNA. Such technology isnow well known in the art, and antisense molecules can be designed atvarious locations along the nucleotide sequences. By treatment of cellsor whole test animals with such antisense sequences, the gene ofinterest is effectively turned off. Frequently, the function of the geneis ascertained by observing behavior at the intracellular, cellular,tissue or organismal level (e.g., lethality, loss of differentiatedfunction, changes in morphology, etc.).

In addition to using sequences constructed to interrupt transcription ofa particular open reading frame, modifications of gene expression isobtained by designing antisense sequences to intron regions,promoter/enhancer elements, or even to transacting regulatory genes.

Example 4 Expression of NMU1

Expression of NMU1 is accomplished by subcloning the cDNAs intoappropriate expression vectors and transfecting the vectors intoexpression hosts such as, e.g., E. coli. In a particular case, thevector is engineered such that it contains a promoter forβ-galactosidase, upstream of the cloning site, followed by sequencecontaining the amino-terminal Methionine and the subsequent sevenresidues of β-galactosidase. Immediately following these eight residuesis an engineered bacteriophage promoter useful for artificial primingand transcription and for providing a number of unique endonucleaserestriction sites for cloning.

Induction of the isolated, transfected bacterial strain withIsopropyl-β-D-thiogalactopyranoside (IPTG) using standard methodsproduces a fusion protein corresponding to the first seven residues ofβ-galactosidase, about 15 residues of “linker”, and the peptide encodedwithin the cDNA. Since cDNA clone inserts are generated by anessentially random process, there is probability of 33% that theincluded cDNA will lie in the correct reading frame for propertranslation. If the cDNA is not in the proper reading frame, it isobtained by deletion or insertion of the appropriate number of basesusing well known methods including in vitro mutagenesis, digestion withexonuclease III or mung bean nuclease, or the inclusion of anoligonucleotide linker of appropriate length.

The NMU1 cDNA is shuttled into other vectors known to be useful forexpression of proteins in specific hosts. Oligonucleotide primerscontaining cloning sites as well as a segment of DNA (about 25 bases)sufficient to hybridize to stretches at both ends of the target cDNA issynthesized chemically by standard methods. These primers are then usedto amplify the desired gene segment by PCR. The resulting gene segmentis digested with appropriate restriction enzymes under standardconditions and isolated by gel electrophoresis. Alternately, similargene segments are produced by digestion of the cDNA with appropriaterestriction enzymes. Using appropriate primers, segments of codingsequence from more than one gene are ligated together and cloned inappropriate vectors. It is possible to optimize expression byconstruction of such chimeric sequences.

Suitable expression hosts for such chimeric molecules include, but arenot limited to, mammalian cells such as Chinese Hamster Ovary (CHO) andhuman 293 cells., insect cells such as Sf9 cells, yeast cells such asSaccharomyces cerevisiae and bacterial cells such as E. coli. For eachof these cell systems, a useful expression vector also includes anorigin of replication to allow propagation in bacteria, and a selectablemarker such as the β-lactamase antibiotic resistance gene to allowplasmid selection in bacteria. In addition, the vector may include asecond selectable marker such as the neomycin phosphotransferase gene toallow selection in transfected eukaryotic host cells. Vectors for use ineukaryotic expression hosts require RNA processing elements such as 3′polyadenylation sequences if such are not part of the cDNA of interest.

Additionally, the vector contains promoters or enhancers which increasegene expression. Such promoters are host specific and include MMTV,SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGHpromoters for yeast. Transcription enhancers, such as the rous sarcomavirus enhancer, are used in mammalian host cells. Once homogeneouscultures of recombinant cells are obtained through standard culturemethods, large quantities of recombinantly produced NMU1 are recoveredfrom the conditioned medium and analyzed using chromatographic methodsknown in the art. For example, NMU1 can be cloned into the expressionvector pcDNA3, as exemplified herein. This product can be used totransform, for example, HEK293 or COS by methodology standard in theart. Specifically, for example, using Lipofectamine (Gibco BRL catologno. 18324-020) mediated gene transfer.

Example 5 Isolation of Recombinant NMU1

NMU1 is expressed as a chimeric protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals [Appa Rao, 1997] and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp., Seattle, Wash.). The inclusion of a cleavable linker sequencesuch as Factor Xa or enterokinase (Invitrogen, Groningen, TheNetherlands) between the purification domain and the NMU1 sequence isuseful to facilitate expression of NMU1.

Example 6 Testing of Chimeric GPCRs

Functional chimeric GPCRs are constructed by combining the extracellularreceptive sequences of a new isoform with the transmembrane andintracellular segments of a known isoform for test purposes. Thisconcept was demonstrated by Kobilka et al. (1988), Science240:1310-1316) who created a series of chimeric α2-β2 adrenergicreceptors (AR) by inserting progressively greater amounts of α2-ARtransmembrane sequence into β2-AR. The binding activity of knownagonists changed as the molecule shifted from having more α2 than β2conformation, and intermediate constructs demonstrated mixedspecificity. The specificity for binding antagonists, however,correlated with the source of the domain VII. The importance of T7Gdomain VII for ligand recognition was also found in chimeras utilizingtwo yeast α-factor receptors and is significant because the yeastreceptors are classified as miscellaneous receptors. Thus, functionalrole of specific domains appears to be preserved throughout the GPCRfamily regardless of category.

In parallel fashion, internal segments or cytoplasmic domains from aparticular isoform are exchanged with the analogous domains of a knownGPCRs and used to identify the structural determinants responsible forcoupling the receptors to trimeric G-proteins. A chimeric receptor inwhich domains V, VI, and the intracellular connecting loop from β2-ARwere substituted into α2-AR was shown to bind ligands with α2-ARspecificity, but to stimulate adenylate cyclase in the manner of β2-AR.This demonstrates that for adrenergic-type receptors, G-proteinrecognition is present in domains V and VI and their connecting loop.The opposite situation was predicted and observed for a chimera in whichthe V->VI loop from α1-AR replaced the corresponding domain on β2-AR andthe resulting receptor bound ligands with β2-AR specificity andactivated G-protein-mediated phosphatidylinositol turnover in the α1-ARmanner. Finally, chimeras constructed from muscarinic receptors alsodemonstrated that V->VI loop is the major determinant for specificity ofG-protein activity.

Chimeric or modified GPCRs containing substitutions in the extracellularand transmembrane regions have shown that these portions of the receptordetermine ligand binding specificity. For example, two Serine residuesconserved in domain V of all adrenergic and D catecholainine GPCRs arenecessary for potent agonist activity. These serines are believed toform hydrogen bonds with the catechol moiety of the agonists within theGPCR binding site. Similarly, an Asp residue present in domain III ofall GPCRs which bind biogenic amines is believed to form an ion pairwith the ligand amine group in the GPCR binding site.

Functional, cloned GPCRs are expressed in heterologous expressionsystems and their biological activity assessed. One heterologous systemintroduces genes for a mammalian GPCR and a mammalian G-protein intoyeast cells. The GPCR is shown to have appropriate ligand specificityand affinity and trigger appropriate biological activation (growtharrest and morphological changes) of the yeast cells.

An alternate procedure for testing chimeric receptors is based on theprocedure utilizing the purinergic receptor (P₂u). Function is easilytested in cultured K562 human leukemia cells because these cells lackP₂u receptors. K562 cells are transfected with expression vectorscontaining either normal or chimeric P₂u and loaded with fura-a,fluorescent probe for Ca⁺⁺. Activation of properly assembled andfunctional P₂u receptors with extracellular UTP or ATP mobilizesintracellular Ca⁺⁺ which reacts with fura-a and is measuredspectrofluorometrically.

As with the GPCRs above, chimeric genes are created by combiningsequences for extracellular receptive segments of any new GPCRpolypeptide with the nucleotides for the transmembrane and intracellularsegments of the known P₂u molecule. Bathing the transfected K562 cellsin microwells containing appropriate ligands triggers binding andfluorescent activity defining effectors of the GPCR molecule. Onceligand and function are established, the P₂u system is useful fordefining antagonists or inhibitors which block binding and prevent suchfluorescent reactions.

Example 7 Production of NMU1 Specific Antibodies

Two approaches are utilized to raise antibodies to NMU1, and eachapproach is useful for generating either polyclonal or monoclonalantibodies. In one approach, denatured protein from reverse phase HPLCseparation is obtained in quantities up to 75 mg. This denatured proteinis used to immunize mice or rabbits using standard protocols; about 100μg are adequate for immunization of a mouse, while up to 1 mg might beused to immunize a rabbit. For identifying mouse hybridomas, thedenatured protein is radioiodinated and used to screen potential murineB-cell hybridomas for those which produce antibody. This procedurerequires only small quantities of protein, such that 20 mg is sufficientfor labeling and screening of several thousand clones.

In the second approach, the amino acid sequence of an appropriate NMU1domain, as deduced from translation of the cDNA, is analyzed todetermine regions of high antigenicity. Oligopeptides comprisingappropriate hydrophilic regions are synthesized and used in suitableimmunization protocols to raise antibodies. The optimal amino acidsequences for immunization are usually at the C-terminus, the N-terminusand those intervening, hydrophilic regions of the polypeptide which arelikely to be exposed to the external environment when the protein is inits natural conformation.

Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH;Sigma, St. Louis, Mo.) by reaction withM-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS. If necessary, acysteine is introduced at the N-terminus of the peptide to permitcoupling to KLH. Rabbits are immunized with the peptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity by binding the peptide to plastic, blocking with 1%bovine serum albumin, reacting with antisera, washing and reacting withlabeled (radioactive or fluorescent), affinity purified, specific goatanti-rabbit IgG.

Hybridomas are prepared and screened using standard techniques.Hybridomas of interest are detected by screening with labeled NMU1 toidentify those fusions producing the monoclonal antibody with thedesired specificity. In a typical protocol, wells of plates (FAST;Becton-Dickinson, Palo Alto, Calif.) are coated during incubation withaffinity purified, specific rabbit anti-mouse (or suitable antispecies 1g) antibodies at 10 mg/ml. The coated wells are blocked with 1% bovineserum albumin, (BSA), washed and incubated with supernatants fromhybridomas. After washing the wells are incubated with labeled NMU1 at 1mg/ml. Supernatants with specific antibodies bind more labeled NMU1 thanis detectable in the background.

Then clones producing specific antibodies are expanded and subjected totwo cycles of cloning at limiting dilution. Cloned hybridomas areinjected into pristane-treated mice to produce ascites, and monoclonalantibody is purified from mouse ascitic fluid by affinity chromatographyon Protein A. Monoclonal antibodies with affinities of at least 10⁸ M⁻¹,preferably 10⁹ to 10¹⁰ M⁻¹ or stronger, are typically made by standardprocedures.

Example 8 Diagnostic Test Using NMU1 Specific Antibodies

Particular NMU1 antibodies are useful for investigating signaltransduction and the diagnosis of infectious or hereditary conditionswhich are characterized by differences in the amount or distribution ofNMU1 or downstream products of an active signaling cascade.

Diagnostic tests for NMU1 include methods utilizing antibody and a labelto detect NMU1 in human body fluids, membranes, cells, tissues orextracts of such. The polypeptides and antibodies of the presentinvention are used with or without modification. Frequently, thepolypeptides and antibodies are labeled by joining them, eithercovalently or noncovalently, with a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and have been reported extensively in both the scientific andpatent literature. Suitable labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent agents, chemiluminescentagents, chromogenic agents, magnetic particles and the like.

A variety of protocols for measuring soluble or membrane-bound NMU1,using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson NMU1 is preferred, but a competitive binding assay may be employed.

Example 9 Purification of Native NMU1 Using Specific Antibodies

Native or recombinant NMU1 is purified by immunoaffinity chromatographyusing antibodies specific for NMU1. In general, an immunoaffinity columnis constructed by covalently coupling the anti-TRH antibody to anactivated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated Sepharose (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such immunoaffinity columns are utilized in the purification of NMU1 bypreparing a fraction from cells containing NMU1 in a soluble form. Thispreparation is derived by solubilization of whole cells or of asubcellular fraction obtained via differential centrifugation (with orwithout addition of detergent) or by other methods well known in theart. Alternatively, soluble NMU1 containing a signal sequence issecreted in useful quantity into the medium in which the cells aregrown.

A soluble NMU1-containing preparation is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of NMU1 (e.g., high ionic strength buffers inthe presence of detergent). Then, the column is eluted under conditionsthat disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or ahigh concentration of a chaotrope such as urea or thiocyanate ion), andNMU1 is collected.

Example 10 Drug Screening

This invention is particularly useful for screening therapeuticcompounds by using NMU1 or binding fragments thereof in any of a varietyof drug screening techniques. As NMU1 is a G protein coupled receptorany of the methods commonly used in the art may potentially be used toidentify NMU1 ligands. For example, the activity of a G protein coupledreceptor such as NMU1 can be measured using any of a variety ofappropriate functional assays in which activation of the receptorresults in an observable change in the level of some second messengersystem, such as adenylate cyclase, guanylylcyclase, calciummobilization, or inositol phospholipid hydrolysis. Alternatively, thepolypeptide or fragment employed in such a test is either free insolution, affixed to a solid support, borne on a cell surface or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. Suchcells, either in viable or fixed form, are used for standard bindingassays.

Measured, for example, is the formation of complexes between NMU1 andthe agent being tested. Alternatively, one examines the diminution incomplex formation between NMU1 and a ligand caused by the agent beingtested.

Thus, the present invention provides methods of screening for drugcanditates, drugs, or any other agents which affect signal transduction.These methods, well known in the art, comprise contacting such an agentwith NMU1 polypeptide or a fragment thereof and assaying (i) for thepresence of a complex between the agent and NMU1 polypeptide orfragment, or (ii) for the presence of a complex between NMU1 polypeptideor fragment and the cell. In such competitive binding assays, the NMU1polypeptide or fragment is typically labeled. After suitable incubation,free NMU1 polypeptide or fragment is separated from that present inbound form, and the amount of free or uncomplexed label is a measure ofthe ability of the particular agent to bind to NMU1 or to interfere withthe NMU1-agent complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to NMU1 polypeptides.Briefly stated, large numbers of different small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with NMU1 polypeptideand washed. Bound NMU1 polypeptide is then detected by methods wellknown in the art. Purified NMU1 are also coated directly onto plates foruse in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies are used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding NMU1specifically compete with a test compound for binding to NMU1polypeptides or fragments thereof. In this manner, the antibodies areused to detect the presence of any peptide which shares one or moreantigenic determinants with NMU1.

Example 11 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, agonists, antagonists, or inhibitors. Any of theseexamples are used to fashion drugs which are more active or stable formsof the polypeptide or which enhance or interfere with the function of apolypeptide in vivo.

In one approach, the three-dimensional structure of a protein ofinterest, or of a protein-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide is gained by modeling based onthe structure of homologous proteins. In both cases, relevant structuralinformation is used to design efficient inhibitors. Useful examples ofrational drug design include molecules which have improved activity orstability or which act as inhibitors, agonists, or antagonists of nativepeptides.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design is based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids isexpected to be an analog of the original receptor. The anti-id is thenused to identify and isolate peptides from banks of chemically orbiologically produced peptides. The isolated peptides then act as thepharmacore.

By virtue of the present invention, sufficient amount of polypeptide aremade available to perform such analytical studies as X-raycrystallography. In addition, knowledge of the NMU1 amino acid sequenceprovided herein provides guidance to those employing computer modelingtechniques in place of or in addition to x-ray crystallography.

Example 12 Identification of Other Members of the Signal TransductionComplex

The inventive purified NMU1 is a research tool for identification,characterization and purification of interacting G or other signaltransduction pathway proteins. Radioactive labels are incorporated intoa selected NMU1 domain by various methods known in the art and used invitro to capture interacting molecules. A preferred method involveslabeling the primary amino groups in NMU1 with ¹²⁵I Bolton-Hunterreagent. This reagent has been used to label various molecules withoutconcomitant loss of biological activity.

Labeled NMU1 is useful as a reagent for the purification of moleculeswith which it interacts. In one embodiment of affinity purification,membrane-bound NMU1 is covalently coupled to a chromatography column.Cell-free extract derived from synovial cells or putative target cellsis passed over the column, and molecules with appropriate affinity bindto NMU1. NMU1-complex is recovered from the column, and the NMU1-bindingligand disassociated and subjected to N-terminal protein sequencing. Theamino acid sequence information is then used to identify the capturedmolecule or to design degenerate oligonucleotide probes for cloning therelevant gene from an appropriate cDNA library.

In an alternate method, antibodies are raised against NMU1, specificallymonoclonal antibodies. The monoclonal antibodies are screened toidentify those which inhibit the binding of labeled NMU1. Thesemonoclonal antibodies are then used therapeutically.

Example 13 Use and Administration of Antibodies, Inhibitors, orAntagonists

Antibodies, inhibitors, or antagonists of NMU1 or other treatments andcompunds that are limiters of signal transduction (LSTs), providedifferent effects when administered therapeutically. LSTs are formulatedin a nontoxic, inert, pharmaceutically acceptable aqueous carrier mediumpreferably at a pH of about 5 to 8, more preferably 6 to 8, although pHmay vary according to the characteristics of the antibody, inhibitor, orantagonist being formulated and the condition to be treated.Characteristics of LSTs include solubility of the molecule, itshalf-life and antigenicity/immunogenicity. These and othercharacteristics aid in defining an effective carrier. Native humanproteins are preferred as LSTs, but organic or synthetic moleculesresulting from drug screens are equally effective in particularsituations.

LSTs are delivered by known routes of administration including but notlimited to topical creams and gels; transmucosal spray and aerosol;transdermal patch and bandage; injectable, intravenous and lavageformulations; and orally administered liquids and pills particularlyformulated to resist stomach acid and enzymes. The particularformulation, exact dosage, and route of administration is determined bythe attending physician and varies according to each specific situation.

Such determinations are made by considering multiple variables such asthe condition to be treated, the LST to be administered, and thepharmacokinetic profile of a particular LST. Additional factors whichare taken into account include severity of the disease state, patient'sage, weight, gender and diet, time and frequency of LST administration,possible combination with other drugs, reaction sensitivities, andtolerance/response to therapy. Long acting LST formulations might beadministered every 3 to 4 days, every week, or once every two weeksdepending on half-life and clearance rate of the particular LST.

Normal dosage amounts vary from 0.1 to 10⁵ μg, up to a total dose ofabout 1 g, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in theliterature; see U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212. Thoseskilled in the art employ different formulations for different LSTs.Administration to cells such as nerve cells necessitates delivery in amanner different from that to other cells such as vascular endothelialcells.

It is contemplated that abnormal signal transduction, trauma, ordiseases which trigger NMU1 activity are treatable with LSTs. Theseconditions or diseases are specifically diagnosed by the tests discussedabove, and such testing should be performed in suspected cases of viral,bacterial or fungal infections, allergic responses, mechanical injuryassociated with trauma, hereditary diseases, lymphoma or carcinoma, orother conditions which activate the genes of lymphoid or neuronaltissues.

Example 14 Production of Non-human Transgenic Animals

Animal model systems which elucidate the physiological and behavioralroles of the NMU1 are produced by creating nonhuman transgenic animalsin which the activity of the NMU1 is either increased or decreased, orthe amino acid sequence of the expressed NMU1 is altered, by a varietyof techniques. Examples of these techniques include, but are not limitedto: 1) Insertion of normal or mutant versions of DNA encoding a NMU1, bymicroinjection, electroporation, retroviral transfection or other meanswell known to those skilled in the art, into appropriately fertilizedembryos in order to produce a transgenic animal or 2) homologousrecombination of mutant or normal, human or animal versions of thesegenes with the native gene locus in transgenic animals to alter theregulation of expression or the structure of these NMU1 sequences. Thetechnique of homologous recombination is well known in the art. Itreplaces the native gene with the inserted gene and hence is useful forproducing an animal that cannot express native NMU1s but does express,for example, an inserted mutant NMU1, which has replaced the native NMU1in the animal's genome by recombination, resulting in under expressionof the transporter. Microinjection adds genes to the genome, but doesnot remove them, and the technique is useful for producing an animalwhich expresses its own and added NMU1, resulting in over expression ofthe NMU1.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as cesiumchloride M2 medium. DNA or cDNAencoding NMU1 is purified from a vector by methods well known to the oneskilled in the art. Inducible promoters may be fused with the codingregion of the DNA to provide an experimental means to regulateexpression of the transgene. Alternatively or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the transgene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a piper puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse which is a mouse stimulated by the appropriate hormones in orderto maintain false pregnancy, where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting DNA into the egg but is used here only forexemplary purposes.

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1. A method of screening for therapeutic agents useful in the treatmentof a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising the steps ofi) contacting a test compound with a NMU1 polypeptide, ii) detectbinding of said test compound to said NMU1 polypeptide.
 2. A method ofscreening for therapeutic agents useful in the treatment of a diseasecomprised in a group of diseases consisting of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver in a mammal comprising the steps of i) determining the activity ofa NMU1 polypeptide at a certain concentration of a test compound or inthe absence of said test compound, ii) determining the activity of saidpolypeptide at a different concentration of said test compound.
 3. Amethod of screening for therapeutic agents useful in the treatment of adisease comprised in a group of diseases consisting of hematologicaldiseases, cardiovascular diseases, disorders of the peripheral andcentral nervous system, inflammation diseases, cancer diseases, anddisorders of the liver in a mammal comprising the steps of i)determining the activity of a NMU1 polypeptide at a certainconcentration of a test compound, ii) determining the activity of a NMU1polypeptide at the presence of a compound known to be a regulator of aNMU1 polypeptide.
 4. The method of any of claims 1 to 3, wherein thestep of contacting is in or at the surface of a cell.
 5. The method ofany of claims 1 to 3, wherein the cell is in vitro.
 6. The method of anyof claims 1 to 3, wherein the step of contacting is in a cell-freesystem.
 7. The method of any of claims 1 to 3, wherein the polypeptideis coupled to a detectable label.
 8. The method of any of claims 1 to 3,wherein the compound is coupled to a detectable label.
 9. The method ofany of claims 1 to 3, wherein the test compound displaces a ligand whichis first bound to the polypeptide.
 10. The method of any of claims 1 to3, wherein the polypeptide is attached to a solid support.
 11. Themethod of any of claims 1 to 3, wherein the compound is attached to asolid support.
 12. A method of screening for therapeutic agents usefulin the treatment of a disease comprised in a group of diseasesconsisting of hematological diseases, cardiovascular diseases, disordersof the peripheral and central nervous system, inflammation diseases,cancer diseases, and disorders of the liver in a mammal comprising thesteps of i) contacting a test compound with a NMU1 polynucleotide, ii)detect binding of said test compound to said NMU1 polynucleotide. 13.The method of claim 12 wherein the nucleic acid molecule is RNA.
 14. Themethod of claim 12 wherein the contacting step is in or at the surfaceof a cell.
 15. The method of claim 12 wherein the contacting step is ina cell-free system.
 16. The method of claim 12 wherein polynucleotide iscoupled to a detectable label.
 17. The method of claim 12 wherein thetest compound is coupled to a detectable label.
 18. A method ofdiagnosing a disease comprised in a group of diseases consisting ofhematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising the steps ofi) determining the amount of a NMU1 polynucleotide in a sample takenfrom said mammal, ii) determining the amount of NMU1 polynucleotide inhealthy and/or diseased mammals.
 19. A pharmaceutical composition forthe treatment of a disease comprised in a group of diseases consistingof hematological diseases, cardiovascular diseases, disorders of theperipheral and central nervous system, inflammation diseases, cancerdiseases, and disorders of the liver in a mammal comprising atherapeutic agent which binds to a NMU1 polypeptide.
 20. Apharmaceutical composition for the treatment of a disease comprised in agroup of diseases consisting of hematological diseases, cardiovasculardiseases, disorders of the peripheral and central nervous system,inflammation diseases, cancer diseases, and disorders of the liver in amammal comprising a therapeutic agent which regulates the activity of aNMU1 polypeptide.
 21. A pharmaceutical composition for the treatment ofa disease comprised in a group of diseases consisting of hematologicaldiseases, cardiovascular diseases, disorders of the peripheral andcentral nervous system, inflammation diseases, cancer diseases, anddisorders of the liver in a mammal comprising a therapeutic agent whichregulates the activity of a NMU1 polypeptide, wherein said therapeuticagent is i) a small molecule, ii) an RNA molecule, iii) an antisenseoligonucleotide, iv) a polypeptide, v) an antibody, or vi) a ribozyme.22. A pharmaceutical composition for the treatment of a diseasecomprised in a group of diseases consisting of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver in a mammal comprising a NMU1 polynucleotide.
 23. A pharmaceuticalcomposition for the treatment of a disease comprised in a group ofdiseases consisting of hematological diseases, cardiovascular diseases,disorders of the peripheral and central nervous system, inflammationdiseases, cancer diseases, and disorders of the liver in a mammalcomprising a NMU1 polypeptide.
 24. Use of regulators of a NMU1 for thepreparation of a pharmaceutical composition for the treatment of adisease comprised in a group of diseases consisting of hematologicaldiseases, cardiovascular diseases, disorders of the peripheral andcentral nervous system, inflammation diseases, cancer diseases, anddisorders of the liver in a mammal.
 25. Method for the preparation of apharmaceutical composition useful for the treatment of a diseasecomprised in a group of diseases consisting of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver in a mammal comprising the steps of i) identifying a regulator ofNMU1, ii) determining whether said regulator ameliorates the symptoms ofa disease comprised in a group of diseases consisting of hematologicaldiseases, cardiovascular diseases, disorders of the peripheral andcentral nervous system, inflammation diseases, cancer diseases, anddisorders of the liver in a mammal; and iii) combining of said regulatorwith an acceptable pharmaceutical carrier.
 26. Use of a regulator ofNMU1 for the regulation of NMU1 activity in a mammal having a diseasecomprised in a group of diseases consisting of hematological diseases,cardiovascular diseases, disorders of the peripheral and central nervoussystem, inflammation diseases, cancer diseases, and disorders of theliver.